Processes for producing fermentation products

ABSTRACT

The present invention relates to processes for producing fermentation products from starch-containing material, wherein an alpha-amylase and a thermostable hemicellulase is present and/or added during liquefaction. The invention also relates to compositions suitable for use in processes of the invention.

FIELD OF THE INVENTION

The present invention relates to processes for producing fermentationproducts, especially ethanol, from starch-containing material. Theinvention also relates to compositions suitable for use in a process ofthe invention and the use of compositions of the invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, fromstarch-containing material is well-known in the art. Industrially twodifferent kinds of processes are used today. The most commonly usedprocess, often referred to as a “conventional process”, includesliquefying gelatinized starch at high temperature using typically abacterial alpha-amylase, followed by simultaneous saccharification andfermentation carried out in the presence of a glucoamylase and afermentation organism. Another well-known process, often referred to asa “raw starch hydrolysis”-process (RSH process), includes simultaneouslysaccharifying and fermenting granular starch below the initialgelatinization temperature typically in the presence of at least aglucoamylase.

Despite significant improvement of fermentation product productionprocesses over the past decade a significant amount of residual starchmaterial is not converted into the desired fermentation product, such asethanol.

Therefore, there is still a desire and need for providing processes forproducing fermentation products, such as ethanol, from starch-containingmaterial that can provide a higher fermentation product yield, or otheradvantages, compared to conventional processes.

SUMMARY OF THE INVENTION

The present invention relates to processes of producing fermentationproducts, such as especially ethanol, from starch-containing materialusing a fermenting organism. The invention also relates to compositionsfor use in processes of the invention.

In the first aspect the invention relates to processes for producingfermentation products, such as preferably ethanol, fromstarch-containing material comprising the steps of:

-   i) liquefying the starch-containing material at a temperature above    the initial gelatinization temperature, preferably between 80-90°    C., using:

an alpha-amylase, such as a bacterial alpha-amylase;

a hemicellulase, preferably a xylanase, having a Melting Point (DSC)above 80° C.;

optionally an endoglucanase having Melting Point (DSC) above 70° C.;

-   ii) saccharifying using a carbohydrate-source generating enzyme;-   iii) fermenting using a fermenting organism.

In a preferred embodiment the hemicellulase is a xylanase, in particulara GH10 xylanase or a GH11 xylanase.

The hemicellulase, in particular a xylanase, especially GH10 or GH11xylanase, preferably has a Melting Point (DSC) above 82° C., such asabove 84° C., such as above 86° C., such as above 88° C., such as above88° C., such as above 90° C., such as above 92° C., such as above 94°C., such as above 96° C., such as above 98° C., such as above 100° C.,such as between 80° C. and 110° C., such as between 82° C. and 110° C.,such as between 84° C. and 110° C.

Examples of suitable hemicellulases, in particular xylanases, includethe xylanase shown in SEQ ID NOs: 3 herein or SEQ ID NO: 34 hereinderived from a strain of Dictyogllomus thermophilum; the xylanase shownin SEQ ID NO: 4 herein derived from a strain of Rasomsoniabyssochlamydoides; the xylanase shown in SEQ ID NO: 5 herein derivedfrom a strain of Talaromyces leycettanus; the xylanase shown in SEQ IDNO: 8 herein derived from a strain of Aspergillus fumigatus or apolypeptide having hemicellulase activity, preferably xylanase activity,having at least 60%, such as at least 70%, such as at least 75%identity, preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 91%, more preferablyat least 92%, even more preferably at least 93%, most preferably atleast 94%, and even most preferably at least 95%, such as even at least96%, at least 97%, at least 98%, at least 99%, such as 100% identity tothe mature part of the polypeptides of SEQ ID NOs: 3, 4, 5, 8 and 34herein.

In another aspect the invention relates to compositions comprising:

-   -   an alpha-amylase;    -   a hemicellulase, in particular a xylanase, having a Melting        Point (DSC) above 80° C., preferably above 85° C., especially        above 90° C., in particular above 95° C.;    -   optionally an endoglucanase;    -   optionally a protease;    -   optionally a carbohydrate-source generating enzyme.

Other enzymes such as pullulanases and phytases may also be comprised inthe composition of the invention.

In a preferred embodiment the composition of the invention comprises

-   -   an alpha-amylase, preferably a bacterial alpha-amylase;    -   a hemicellulase having a Melting Point (DSC) above 80° C.,        preferably above 85° C., especially above 90° C., in particular        above 95° C.;    -   an endoglucanase having Melting Point (DSC) above 70° C.,        preferably above 75° C., especially above 80° C., in particular        above 85° C.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the ethanol yield with TI EG (SEQ ID NO: 9) and TI xylanase(SEQ ID NO: 5) or Dt xylanase (SEQ ID NO: 34) addition in liquefaction,and SSF with Glucoamylase SA (GSA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to processes of producing fermentationproducts, such as ethanol, from starch-containing material using afermenting organism. The invention also relates to compositions for usein processes of the invention.

The inventors have found that increased ethanol yield is obtained whenliquefying starch-containing material using an alpha-amylase in thepresence of a thermostable hemicellulase (see Example 19). It was alsofound that when additionally adding a thermostable endoglucase theethanol yield increased even more.

In the first aspect the invention relates to processes for producingfermentation products, such as preferably ethanol, fromstarch-containing material comprising the steps of:

-   i) liquefying the starch-containing material at a temperature above    the initial gelatinization temperature, such as between 80-90° C.,    using:

an alpha-amylase, such as a bacterial alpha-amylase;

a hemicellulase having a Melting Point (DSC) above 80° C.;

-   ii) saccharifying using a carbohydrate-source generating enzyme;-   iii) fermenting using a fermenting organism.

Steps ii) and iii) may be carried out either sequentially orsimultaneously. In a preferred embodiment steps ii) and iii) are carriedout simultaneously. The alpha-amylase, the hemicellulase, preferablyxylanase, having a Melting Point (DSC) above 80° C.; and optionally athermostable endoglucanase having a Melting Point (DSC) above 70° C.,may be added before and/or during liquefaction step i). Optionally aprotease, a carbohydrate-source generating enzyme, preferably aglucoamylase, a pullulanase, and/or a phytase may be present and/oradded as well. In a preferred embodiment a composition of the inventiondefined below may suitably be used in liquefaction in a process of theinvention. The enzymes may be added individually or as one or more blendcompositions comprising an alpha-amylase, hemicellulase, preferablyxylanase, having a Melting Point (DSC) above 80° C., and optionalendoglucanase and optionally a protease, a carbohydrate-sourcegenerating enzyme, a pullulanase and/or a phytase.

Examples of alpha-amylases can be found in the “Alpha-Amylase Presentand/or Added During Liquefaction”-section below.

In an embodiment the alpha-amylase is a variant of the alpha-amylaseshown in SEQ ID NO: 1 herein, such as one derived from a strain Bacillusstearomthermphilus, with mutations selected from the group of:

-   I181*+G182*;-   I181*+G182*+N193F;-   preferably-   I181*+G182*+E129V+K177L+R179E;-   I181*+G182*+N193F+E129V+K177L+R179E:-   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;-   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; and-   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using    SEQ ID NO: 1 herein for numbering).

Bacillus stearothermophilus alpha-amylases are typically naturallytruncated when produced to be around 491 amino acids long (compared toSEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein), such as from about480-495 amino acids long.

In an embodiment the bacterial alpha-amylase, e.g., Bacillusalpha-amylase, such as especially Bacillus stearothermophilusalpha-amylase, is dosed in liquefaction in a concentration between0.01-10 KNU-A/g DS, e.g., between 0.02 and 5 KNU-A/g DS, such as 0.03and 3 KNU-A, preferably 0.04 and 2 KNU-A/g DS, such as especially 0.01and 2 KNU-A/g DS.

In an embodiment the bacterial alpha-amylase, e.g., Bacillusalpha-amylase, such as especially Bacillus stearothermophilusalpha-amylase, is dosed in liquefaction in a concentration of between0.0001-1 mg EP (Enzyme Protein)/g DS, e.g., 0.0005-0.5 mg EP/g DS, suchas 0.001-0.1 mg EP/g DS.

Examples of suitable hemicellulases, such as xylanases, having a MeltingPoint (DSC) above 80° C. can be found in the “ThermostableHemicellulases Present and/or Added During Liquefaction”-section below.

Specific examples of suitable hemicellulases, in particular xylanases,include the xylanases shown in SEQ ID NOs: 3 herein and SEQ ID NO: 34herein, e.g., derived from a strain of Dictyogilomus thermophilum; thexylanase shown in SEQ ID NO: 4 herein, e.g., derived from a strain ofRasomsonia byssochiamydoides; the xylanase shown in SEQ ID NO: 5 herein,e.g., derived from a strain of Talaromyces leycettanus; the xylanaseshown in SEQ ID NO: 8 herein, e.g., derived from a strain of Aspergillusfumigatus or polypeptides having hemicellulase, preferably xylanaseactivity, having at least 60%, such as at least 70%, such as at least75% identity, preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, such as100% identity to the mature part of any of the polypeptides of SEQ IDNOs: 3, 4, 5, 8 and 34 herein, respectively.

Examples of suitable optional endoglucanases having a Melting Point(DSC) above 70° C. can be found in the “Thermostable EndoglucanasePresent and/or Added During Liquefaction”-section below.

In a preferred embodiment the endoglucanase is the one shown in SEQ IDNO: 9 herein, such as one derived from a strain of Talaromycesleycettanus (WO2013/019780), or an endoglucanase having at least 80%identity to SEQ ID NO: 9 herein.

In a preferred embodiment the endoglucanase is the one shown in SEQ IDNO: 9 herein, such as one derived from a strain of Talaromycesleycettanus (WO2013/019780—hereby incorporated by reference), or anendoglucanase having at least 90% identity to SEQ ID NO: 9 herein havinga Melting Point (DSC) above 70° C.

Examples of optional proteases can be found in the “Protease Presentand/or Added During Liquefaction”-section below.

Examples of suitable optional carbohydrate-source generating enzymes,preferably thermostable carbohydrate-source generating enzymes, inparticularglucoamylases, can be found in the “Carbohydrate-SourceGenerating Enzymes Present and/or Added During Liquefaction”-sectionbelow.

A suitable optional pullulanase can be found in the “Pullulanase Presentand/or Added During Liquefaction”-section below. In a preferredembodiment the pullulanase is derived from Bacillus sp.

Examples of optional phytases can be found in the “Phytase Presentand/or Added During Liquefaction”-section below. In a preferredembodiment the phytase is derived from a strain of Buttiauxella.

A suitable cellulase or cellulolytic enzyme composition present and/oradded during saccharification and/or fermentation or simultaneoussaccharification and fermentation (SSF) can be found in the “Cellulaseor Cellulolytic Enzyme Composition Present and/or Added DuringSaccharification and/or Fermentation or SSF”-section below. In anembodiment the cellulase or cellulolytic enzyme composition is derivedfrom Trichoderma reesei.

In a preferred embodiment the cellulase or cellulolytic enzymecomposition is a Trichoderma reesei cellulolytic enzyme composition,further comprising Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656 or SEQID NO: 30 herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO:2 of WO 2005/047499 or SEQ ID NO: 29 herein).

In an embodiment the cellulase or cellulolytic enzyme composition is aTrichoderma reesei cellulolytic enzyme composition further comprisingPenicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO2011/041397 or SEQ ID NO: 31 herein, and Aspergillus fumigatusbeta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ IDNO: 29 herein, or a variant thereof, which variant has one of,preferably all of, the following substitutions: F100D, S283G, N456E,F512Y (using SEQ ID NO: 29 herein for numbering).

According to the process of the invention the pH during liquefaction maybe between 4.0-6.5, such as 4.5-6.2, such as above 4.8-6.0, such asbetween 5.0-5.8.

According to the invention the temperature is above the initialgelatinization temperature. The term “initial gelatinizationtemperature” refers to the lowest temperature at which solubilization ofstarch, typically by heating, begins. The temperature can vary fordifferent starches. The initial gelanitization temperature may be from50-70° C.

In an embodiment the temperature during liquefaction step i) is in therange from 70-100° C., such as between 70-95° C., such as between 75-90°C., preferably between 80-90° C., such as around 85° C.

In an embodiment, the process of the invention further comprises, priorto the step i), the steps of:

a) reducing the particle size of the starch-containing material,preferably by dry milling;

b) forming a slurry comprising the starch-containing material and water.

The starch-containing starting material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure, to increase the surface area and allowing for furtherprocessing. Generally, there are two types of processes: wet and drymilling. In dry milling whole kernels are milled and used. Wet millinggives a good separation of germ and meal (starch granules and protein).Wet milling is often applied at locations where the starch hydrolysateis used in production of, e.g., syrups. Both dry and wet millings arewell known in the art of starch processing. According to the presentinvention dry milling is preferred. The particle size may be reducedeven further, for example, by Turkish grinding. In an embodiment theparticle size is reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5mm, or so that at least 30%, preferably at least 50%, more preferably atleast 70%, even more preferably at least 90% of the starch-containingmaterial fit through a sieve with a 0.05 to 3.0 mm screen, preferably0.1-0.5 mm screen, In another embodiment at least 50%, preferably atleast 70%, more preferably at least 80%, especially at least 90% of thestarch-containing material fit through a sieve with #6 screen. In anembodiment, at least 75% of the starch-containing material fit through asieve with less than a 0.5 mm screen, more preferably at least 79% or80% of the starch-containing material fit through a sieve with less thana 0.425 mm screen. In an embodiment, at least 50% of thestarch-containing material fit through a sieve with a 0.25 to 0.425 mmscreen, more preferably at least 59% or 60% of the starch-containingmaterial fit through a sieve with a 0.25 to 0.425 mm screen.

The aqueous slurry may contain from 10-55 w/w-% dry solids (DS),preferably 25-45 w/w-% dry solids (DS), more preferably 30-40 w/w-% drysolids (DS) of starch-containing material.

The slurry may be heated to above the initial gelatinizationtemperature, preferably to between 70-95° C., such as between 80-90° C.,between pH 5.0-7.0, preferably between 5.0 and 6.0, for 30 minutes to 5hours, such as around 2 hours.

In an embodiment liquefaction step i) is carried out for 0.5-5 hours ata temperature from 70-95° C. at a pH from 4-6.

In a preferred embodiment liquefaction step i) is carried out for 0.5-3hours at a temperature from 80-90° C. at a pH from 4-6.

The alpha-amylase and hemicellulase and optional thermostableendoglucanase, optional protease, optional carbohydrate-sourcegenerating enzyme, in particular glucoamylase, optional pullulanase,and/or optional phytase, may initially be added to the aqueous slurry toinitiate liquefaction (thinning). In an embodiment only a portion of theenzymes is added to the aqueous slurry, while the rest of the enzymesare added during liquefaction step i).

The aqueous slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to liquefaction in step i).The jet-cooking may be carried out at a temperature between 95-160° C.,such as between 110-145° C., preferably 120-140° C., such as 125-135°C., preferably around 130° C. for about 1-15 minutes, preferably forabout 3-10 minutes, especially around about 5 minutes.

Saccharification and Fermentation

According to the process of the invention one or morecarbohydrate-source generating enzymes, in particular glucoamylase, maybe present and/or added during saccharification step ii) and/orfermentation step iii). The carbohydrate-source generating enzyme maypreferably be a glucoamylase, but may also be an enzyme selected fromthe group consisting of: beta-amylase, maltogenic amylase andalpha-glucosidase. The carbohydrate-source generating enzyme addedduring saccharification step ii) and/or fermentation step iii) istypically different from the optional carbohydrate-source generatingenzyme, in particular glucoamylase, optionally added during liquefactionstep i). In an embodiment the carbohydrate-source generating enzymes, inparticular glucoamylase, is added together with a fungal alpha-amylase.

Examples of carbohydrate-source generating enzymes, includingglucoamylases, can be found in the “Carbohydrate-Source GeneratingEnzyme Present and/or Added During Saccharification and/orFermentation”-section below.

When doing sequential saccharification and fermentation,saccharification step ii) may be carried out at conditions well-known inthe art. For instance, the saccharification step ii) may last up to fromabout 24 to about 72 hours.

In an embodiment a pre-saccharification step is done. In an embodiment acarbohydrate-source generating enzyme is added duringpre-saccharification carried out before saccharification step ii) and/orfermentation step iii). The carbohydrate-source generating enzyme mayalso be added during pre-saccharification carried out beforesimultaneous saccharification and fermentation (SSF).

In an embodiment a carbohydrate-source generating enzyme, preferablyglucoamylase, and/or the cellulase or cellulolytic enzyme composition,are added during pre-saccharification carried out beforesaccharification step ii) and/or fermentation step iii). Thecarbohydrate-source generating enzyme, preferably glucoamylase, and thecellulase or cellulolytic enzyme composition may also be added duringpre-saccharification carried out before simultaneous saccharificationand fermentation (SSF). Pre-saccharification is typically done for 40-90minutes at a temperature between 30-65° C., typically around 60° C.Pre-saccharification may be followed by saccharification duringfermentation in simultaneous saccharification and fermentation (“SSF).Saccharification is typically carried out at temperatures from 20-75°C., preferably from 40-70° C., typically around 60° C., and at a pHbetween 4 and 5, such as around pH 4.5.

Simultaneous saccharification and fermentation (“SSE”) is widely used inindustrial scale fermentation product production processes, especiallyethanol production processes. When doing SSF the saccharification stepii) and the fermentation step iii) are carried out simultaneously. Theremay be no holding stage for the saccharification, meaning that afermenting organism, such as yeast, and enzyme(s), in a preferredembodiment according to the invention a glucoamylase and a cellulase orcellulolytic enzyme composition, may be added together. However, it isalso contemplated to add the fermenting organism and enzyme(s)separately. Fermentation or SSF may, according to the invention,typically be carried out at a temperature from 25° C. to 40° C., such asfrom 28° C. to 35° C., such as from 30° C. to 34° C., preferably aroundabout 32° C. In an embodiment fermentation is ongoing for 6 to 120hours, in particular 24 to 96 hours. In an embodiment the pH is between3.5-5, in particular between 3.8 and 4.3.

Fermentation Medium

“Fermentation media” or “fermentation medium” refers to the environmentin which fermentation is carried out. The fermentation medium includesthe fermentation substrate, that is, the carbohydrate source that ismetabolized by the fermenting organism. According to the invention thefermentation medium may comprise nutrients and growth stimulator(s) forthe fermenting organism(s). Nutrient and growth stimulators are widelyused in the art of fermentation and include nitrogen sources, such asammonia; urea, vitamins and minerals, or combinations thereof.

Fermenting Organisms

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, especially yeast, suitable for use in afermentation process and capable of producing the desired fermentationproduct. Especially suitable fermenting organisms are able to ferment,i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product, such as ethanol.Examples of fermenting organisms include fungal organisms, such asyeast. Preferred yeast includes strains of Saccharomyces spp., inparticular, Saccharomyces cerevisiae.

Suitable concentrations of the viable fermenting organism duringfermentation, such as SSF, are well known in the art or can easily bedetermined by the skilled person in the art. In one embodiment thefermenting organism, such as ethanol fermenting yeast, (e.g.,Saccharomyces cerevisiae) is added to the fermentation medium so thatthe viable fermenting organism, such as yeast, count per mL offermentation medium is in the range from 10⁵ to 10¹², preferably from10⁷ to 10¹⁰, especially about 5×10⁷.

Examples of commercially available yeast includes, e.g., RED STAR™ andETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI(available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Starch-Containing Materials

Any suitable starch-containing material may be used according to thepresent invention. The starting material is generally selected based onthe desired fermentation product. Examples of starch-containingmaterials, suitable for use in a process of the invention, include wholegrains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum,rice, peas, beans, or sweet potatoes, or mixtures thereof or starchesderived therefrom, or cereals. Contemplated are also waxy and non-waxytypes of corn and barley.

In a preferred embodiment the starch-containing material, used forethanol production according to the invention, is corn or wheat.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation step using a fermenting organism. Fermentationproducts contemplated according to the invention include alcohols (e.g.,ethanol, methanol, butanol; polyols such as glycerol, sorbitol andinositol); organic acids (e.g., citric acid, acetic acid, itaconic acid,lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics(e.g., penicillin and tetracycline); enzymes; vitamins (e.g.,riboflavin, B₁₂, beta-carotene); and hormones. In a preferred embodimentthe fermentation product is ethanol, e.g., fuel ethanol; drinkingethanol, i.e., potable neutral spirits; or industrial ethanol orproducts used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry andtobacco industry. Preferred beer types comprise ales, stouts, porters,lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcoholbeer, low-calorie beer or light beer. Preferably processes of theinvention are used for producing an alcohol, such as ethanol. Thefermentation product, such as ethanol, obtained according to theinvention, may be used as fuel, which is typically blended withgasoline. However, in the case of ethanol it may also be used as potableethanol.

Recovery

Subsequent to fermentation, or SSF, the fermentation product may beseparated from the fermentation medium. The slurry may be distilled toextract the desired fermentation product (e.g., ethanol). Alternativelythe desired fermentation product may be extracted from the fermentationmedium by micro or membrane filtration techniques. The fermentationproduct may also be recovered by stripping or other method well known inthe art.

Alpha-Amylase Present and/or Added During Liquefaction

According to the invention an alpha-amylase is present and/or added inliquefaction together with a hemicellulase, preferably xylanase, havinga Melting Point (DSC) above 80° C., such as between 80° C. and 95° C.,and an optional endoglucanase, an optional protease, an optionalcarbohydrate-source generating enzyme, in particular a glucoamylase, anoptional pullulanase, and/or an optional phytase.

The alpha-amylase added during liquefaction step i) may be anyalpha-amylase. Preferred are bacterial alpha-amylases, such asespecially Bacillus alpha-amylases, such as Bacillus stearothermophilusalpha-amylases, which are stable at temperature used duringliquefaction.

Bacterial Alpha-Amylase

The term “bacterial alpha-amylase” means any bacterial alpha-amylaseclassified under EC 3.2.1.1. A bacterial alpha-amylase used according tothe invention may, e.g., be derived from a strain of the genus Bacillus,which is sometimes also referred to as the genus Geobacillus. In anembodiment the Bacillus alpha-amylase is derived from a strain ofBacillus amyloliquefaciens, Bacillus licheniformis, Bacillusstearothermophilus, Bacillus sp. TS-23, or Bacillus subtilis, but mayalso be derived from other Bacillus sp.

Specific examples of bacterial alpha-amylases include the Bacillusstearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQID NO: 1 herein, the Bacillus amyloliquefaciens alpha-amylase of SEQ IDNO: 5 in WO 99/19467, and the Bacillus licheniformis alpha-amylase ofSEQ ID NO: 4 in WO 99/19467 and the Bacillus sp. TS-23 alpha-amylasedisclosed as SEQ ID NO: 1 in WO 2009/061380 (all sequences are herebyincorporated by reference).

In an embodiment the bacterial alpha-amylase may be an enzyme having adegree of identity of at least 60%, e.g., at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% to any of the sequences shown in SEQ ID NOS: 3, 4 or 5,respectively, in WO 99/19467 and SEQ ID NO: 1 in WO 2009/061380.

In an embodiment the alpha-amylase may be an enzyme having a degree ofidentity of at least 60%, e.g., at least 70%, at least 80%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% to any ofthe sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1herein.

In a preferred embodiment the alpha-amylase is derived from Bacillusstearothermophilus. The Bacillus stearothermophilus alpha-amylase may bea mature wild-type or a mature variant thereof. The mature Bacillusstearothermophilus alpha-amylases, or variant thereof, may be naturallytruncated during recombinant production. For instance, the matureBacillus stearothermophilus alpha-amylase may be truncated at theC-terminal so it is around 491 amino acids long (compared to SEQ ID NO:3 in WO 99/19467 or SEQ ID NO: 1 herein), such as from 480-495 aminoacids long.

The Bacillus alpha-amylase may also be a variant and/or hybrid. Examplesof such a variant can be found in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, WO 02/10355 and WO2009/061380 (alldocuments are hereby incorporated by reference). Specific alpha-amylasevariants are disclosed in U.S. Pat. Nos. 6,093,562, 6,187,576,6,297,038, and 7,713,723 (hereby incorporated by reference) and includeBacillus stearothermophilus alpha-amylase (often referred to as BSGalpha-amylase) variants having a deletion of one or two amino acids atany of positions R179, G180, I181 and/or G182, preferably the doubledeletion disclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (herebyincorporated by reference), preferably corresponding to deletion ofpositions I181 and G182 compared to the amino acid sequence of Bacillusstearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed inWO 99/19467 or SEQ ID NO: 1 herein or the deletion of amino acids R179and G180 using SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein. Evenmore preferred are Bacillus alpha-amylases, especially Bacillusstearothermophilus (BSG) alpha-amylases, which have at one or two aminoacid deletions corresponding to positions R179, G180, I181 and G182,preferably which have a double deletion corresponding to R179 and G180,or preferably a deletion of positions 181 and 182 (denoted I181*+G182*),and optionally further comprises a N193F substitution (denotedI181*+G182*+N193F) compared to the wild-type BSG alpha-amylase aminoacid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or SEQID NO: 1 herein. The bacterial alpha-amylase may also have asubstitution in a position corresponding to S239 in the Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, or aS242 variant in the Bacillus stearothermophilus alpha-amylase of SEQ IDNO: 3 in WO 99/19467 or SEQ ID NO: 1 herein.

In an embodiment the variant is a 5242A, E or Q variant, preferably aS242Q or A variant, of the Bacillus stearothermophilus alpha-amylase(using SEQ ID NO: 1 herein for numbering).

In an embodiment the variant is a position E188 variant, preferablyE188P variant of the Bacillus stearothermophilus alpha-amylase (usingSEQ ID NO: 1 herein for numbering).

Other contemplated variant are Bacillus sp. TS-23 variant disclosed inWO2009/061380, especially variants defined in claim 1 of WO2009/061380(hereby incorporated by reference).

Bacterial Hybrid Alpha-Amylases

The bacterial alpha-amylase may also be a hybrid bacterialalpha-amylase, e.g., an alpha-amylase comprising 445 C-terminal aminoacid residues of the Bacillus licheniformis alpha-amylase (shown in SEQID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues ofthe alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQID NO: 5 of WO 99/19467). In a preferred embodiment this hybrid has oneor more, especially all, of the following substitutions:

-   G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the    Bacillus licheniformis numbering in SEQ ID NO: 4 of WO 99/19467).    Also preferred are variants having one or more of the following    mutations (or corresponding mutations in other Bacillus    alpha-amylases): H154Y, A181T, N190F, A209V and Q264S and/or the    deletion of two residues between positions 176 and 179, preferably    the deletion of E178 and G179 (using SEQ ID NO: 5 of WO 99/19467 for    position numbering).

In an embodiment the bacterial alpha-amylase is the mature part of thechimeric alpha-amylase disclosed in Richardson et al., 2002, The Journalof Biological Chemistry 277(29): 267501-26507, referred to as BD5088 ora variant thereof. This alpha-amylase is the same as the one shown inSEQ ID NO: 2 in WO 2007134207. The mature enzyme sequence starts afterthe initial “Met” amino acid in position 1.

Thermostable Alpha-Amylase

According to the invention the alpha-amylase is used in combination witha hemicellulase, preferably xylanase, having a Melting Point (DSC) above80° C. Optionally an endoglucanase having a Melting Point (DSC) above70° C., such as above 75° C., in particular above 80° C. may beincluded. The thermostable alpha-amylase, such as a bacterial analpha-amylase, is preferably derived from Bacillus stearothermophilus orBacillus sp. TS-23. In an embodiment the alpha-amylase has a T½ (min) atpH 4.5, 85° C., 0.12 mM CaCl₂ of at least 10.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, of at least 15.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, of at least 20.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, of at least 25.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, of at least 30.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, of at least 40.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, of at least 50.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, of at least 60.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 10-70.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 15-70.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 20-70.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 25-70.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 30-70.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 40-70.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 50-70.

In an embodiment the alpha-amylase has a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂, between 60-70.

In an embodiment the alpha-amylase is a bacterial alpha-amylase,preferably derived from the genus Bacillus, especially a strain ofBacillus stearothermophilus, in particular the Bacillusstearothermophilus as disclosed in WO 99/19467 as SEQ ID NO: 3 or SEQ IDNO: 1 herein with one or two amino acids deleted at positions R179,G180, I181 and/or G182, in particular with R179 and G180 deleted, orwith I181 and G182 deleted, with mutations in below list of mutations.In preferred embodiments the Bacillus stearothermophilus alpha-amylaseshave double deletion I181+G182, and optional substitution N193F,optionally further comprising mutations selected from below list:

V59A + Q89R + G112D + E129V + K177L + R179E + K220P + N224L + Q254S;V59A + Q89R + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S;V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E +D281N; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +I270L; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +H274K; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +Y276F; V59A + E129V + R157Y + K177L + R179E + K220P + N224L + S242Q +Q254S; V59A + E129V + K177L + R179E + H208Y + K220P + N224L + S242Q +Q254S; V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + Y276F;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + D281N;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + M284T;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + G416V;V59A + E129V + K177L + R179E + K220P + N224L + Q254S; V59A + E129V +K177L + R179E + K220P + N224L + Q254S + M284T; A91L + M96I + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S; E129V + K177L + R179E;E129V + K177L + R179E + K220P + N224L + S242Q + Q254S; E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + Y276F + L427M; E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + M284T; E129V + K177L + R179E +K220P + N224L + S242Q + Q254S + N376* + I377*; E129V + K177L + R179E +K220P + N224L + Q254S; E129V + K177L + R179E + K220P + N224L + Q254S +M284T; E129V + K177L + R179E + S242Q; E129V + K177L + R179V + K220P +N224L + S242Q + Q254S; K220P + N224L + S242Q + Q254S; M284V; V59A +Q89R + E129V + K177L + R179E + Q254S + M284V.

In an embodiment the alpha-amylase is selected from the group ofBacillus stearomthermphilus alpha-amylase variants:

-   I181w+G182*;-   I181*+G182*+N193F;-   preferably-   I181*+G182*+E129V+K177L+R179E;-   I181*+G182*+N193F+E129V+K177L+R179E;-   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;-   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; and-   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using    SEQ ID NO: 1 herein for numbering).

In an embodiment the bacterial alpha-amylase, such as Bacillusalpha-amylase, such as as Bacillus stearomthermphilus alpha-amylase hasat least 60%, such as at least 70%, such as at least 75% identity,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, such as 100% identity to themature part of the polypeptide of SEQ ID NO: 1 herein.

In an embodiment the bacterial alpha-amylase variant, such as Bacillusalpha-amylase variant, such as Bacillus stearomthermphilus alpha-amylasevariant has at least 60%, such as at least 70%, such as at least 75%identity, preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 91%, more preferablyat least 92%, even more preferably at least 93%, most preferably atleast 94%, and even most preferably at least 95%, such as even at least96%, at least 97%, at least 98%, at least 99%, but less than 100%identity to the mature part of the polypeptide of SEQ ID NO: 1 herein.

It should be understood that when referring to Bacillusstearothermophilus alpha-amylase and variants thereof they are normallyproduced naturally in truncated form. In particular, the truncation maybe so that the Bacillus stearothermophilus alpha-amylase shown in SEQ IDNO: 3 in WO 99/19467 or SEQ ID NO: 1 herein, or variants thereof, aretruncated in the C-terminal and are typically around 491 amino acidslong, such as from 480-495 amino acids long.

Thermostable Hemicellulase Present and/or Added During Liquefaction

According to the invention a hemicellulase, preferably xylanase, havinga Melting Point (DSC) above 80° C. is present and/or added toliquefaction step i) in combination with an alpha-amylase, such as abacterial alpha-amylase (described above).

The thermostability of a hemicellulase, preferably xylanase may bedetermined as described in the “Materials & Methods”-section under“Determination of T_(d) by Differential Scanning calorimetry forEndoglucanases and Hemicellulases”.

In an embodiment the hemicellulase, in particular xylanase, especiallyGH10 or GH11 xylanase has a Melting Point (DSC) above 82° C., such asabove 84° C., such as above 86° C., such as above 88° C., such as above88° C., such as above 90° C., such as above 92° C., such as above 94°C., such as above 96° C., such as above 98° C., such as above 100° C.,such as between 80° C. and 110° C., such as between 82° C. and 110° C.,such as between 84° C. and 110° C.

In a preferred embodiment the hemicellulase, in particular xylanase,especially GH10 xylanase has at least 60%, such as at least 70%, such asat least 75%, preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, such as100% identity to the mature part of the polypeptide of SEQ ID NO: 3herein, preferably derived from a strain of the genus Dictyoglomus, suchas a strain of Dictyogllomus thermophilum.

In a preferred embodiment the hemicellulase, in particular xylanase,especially GH11 xylanase has at least 60%, such as at least 70%, such asat least 75%, preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, such as100% identity to the mature part of the polypeptide of SEQ ID NO: 34herein, preferably derived from a strain of the genus Dictyoglomus, suchas a strain of Dictyogitomus thermophlium.

In a preferred embodiment the hemicellulase, in particular xylanase,especially GH10 xylanase has at least 60%, such as at least 70%, such asat least 75% identity, preferably at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, such as100% identity to the mature part of the polypeptide of SEQ ID NO: 4herein, preferably derived from a strain of the genus Rasamsonia, suchas a strain of Rasomsonia byssochiamydoides.

In a preferred embodiment the hemicellulase, in particular xylanase,especially GH10 xylanase has at least 60%, such as at least 70%, such asat least 75% identity, preferably at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, such as100% identity to the mature part of the polypeptide of SEQ ID NO: 5herein, preferably derived from a strain of the genus Talaromyces, suchas a strain of Talaromyces leycettanus.

In a preferred embodiment the hemicellulase, in particular xylanase,especially GH10 xylanase has at least 60%, such as at least 70%, such asat least 75% identity, preferably at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, such as100% identity to the mature part of the polypeptide of SEQ ID NO: 8herein, preferably derived from a strain of the genus Aspergillus, suchas a strain of Aspergillus fumigatus.

Thermostable Endoglucanase Present and/or Added During Liquefaction

According to the invention an optional endoglucanase (“EG”) having aMelting Point (DSC) above 70° C., such as between 70° C. and 95° C. maybe present and/or added in liquefaction step i) in combination with analpha-amylase, such as a thermostable bacterial alpha-amylase and ahemicellulase, preferably xylanase, having a Melting Point (DSC) above80° C.

The thermostability of an endoglucanase may be determined as describedin the “Materials & Methods”—section under “Determination of T_(d) byDifferential Scanning calorimetry for Endoglucanases andHemicellulases”.

In an embodiment the endoglucanase has a Melting Point (DSC) above 72°C., such as above 74° C., such as above 76° C., such as above 78° C.,such as above 80° C., such as above 82° C., such as above 84° C., suchas above 86° C., such as above 88° C., such as between 70° C. and 95°C., such as between 76° C. and 94° C., such as between 78° C. and 93°C., such as between 80° C. and 92° C., such as between 82° C. and 91°C., such as between 84° C. and 90° C.

In a preferred embodiment the endogluconase used in a process of theinvention or comprised in a composition of the invention is a GlycosideHydrolase Family 5 endoglucnase or GH5 endoglucanase (see the CAZydatabase on the “www.cazy.org” webpage.

In an embodiment the GH5 endoglucanase is from family EG II, such as theTaiaromyces ieycettanus endoglucanase shown in SEQ ID NO: 9 herein;Penicillium capsulatum endoglucanase shown in SEQ ID NO: 35 herein, andTrichophaea saccata endoglucanase shown in SEQ ID NO: 36 herein.

In an embodiment the endoglucanase is a family GH45 endoglucanase. In anembodiment the GH45 endoglucanase is from family EG V, such as theSordaria fimicola shown in SEQ ID NO: 38 herein or the Thielaviaterrestris endoglucanase shown in SEQ ID NO: 37 herein.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 9 herein. In an embodiment the endoglucanase is derivedfrom a strain of the genus Talaromyces, such as a strain of Talaromycesleycettanus.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 35 herein, preferably derived from a strain of the genusPenicillium, such as a strain of Penicillium capsulatum.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 36 herein, preferably derived from a strain of the genusTrichophaea, such as a strain of Trichophaea saccata.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 37 herein, preferably derived from a strain of the genusThielavia, such as a strain of Thielavia terrestris.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 38 herein, preferably derived from a strain of the genusSordaria, such as a strain of Sordaria fimicola.

In an embodiment the endoglucanase is added in liquefaction step i) at adose from 1-10,000 μg EP (Enzymes Protein)/g DS), such as 10-1,000 μgEP/g DS.

Protease Present and/or Added During Liquefaction

In an embodiment of the invention an optional protease, such as athermostable protease, may be present and/or added in liquefactiontogether with an alpha-amylase, such as a thermostable alpha-amylase,and a hemicellulase, preferably xylanase, having a melting point (DSC)above 80° C., and optionally an endoglucanase, a carbohydrate-sourcegenerating enzyme, in particular a glucoamylase, optionally apullulanase and/or optionally a phytase.

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

In a preferred embodiment the thermostable protease used according tothe invention is a “metallo protease” defined as a protease belonging toEC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metalloproteinases).

To determine whether a given protease is a metallo protease or not,reference is made to the above “Handbook of Proteolytic Enzymes” and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any suitable assay, in which asubstrate is employed, that includes peptide bonds relevant for thespecificity of the protease in question. Assay-pH and assay-temperatureare likewise to be adapted to the protease in question. Examples ofassay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples ofassay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80° C.

Examples of protease substrates are casein, such as Azurine-CrosslinkedCasein (AZCL-casein). Two protease assays are described below in the“Materials & Methods”-section, of which the so-called “AZCL-CaseinAssay” is the preferred assay.

In an embodiment the thermostable protease has at least 20%, such as atleast 30%, such as at least 40%, such as at least 50%, such as at least60%, such as at least 70%, such as at least 80%, such as at least 90%,such as at least 95%, such as at least 100% of the protease activity ofthe JTP196 variant (Example 2) or Protease Pfu (SEQ ID NO: 13 herein)determined by the AZCL-casein assay described in the “Materials &Methods”-section.

There are no limitations on the origin of the thermostable protease usedin a process or composition of the invention as long as it fulfills thethermostability properties defined below.

In one embodiment the protease is of fungal origin.

In a preferred embodiment the thermostable protease is a variant of ametallo protease as defined above. In an embodiment the thermostableprotease used in a process or composition of the invention is of fungalorigin, such as a fungal metallo protease, such as a fungal metalloprotease derived from a strain of the genus Thermoascus, preferably astrain of Thermoascus aurantiacus, especially Thermoascus aurantiacusCGMCC No. 0670 (classified as EC 3.4.24.39).

In an embodiment the thermostable protease is a variant of the maturepart of the metallo protease shown in SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 andshown as SEQ ID NO: 2 herein further with mutations selected from belowlist:

S5*+D79L+S87P+A112P+D142L;

D79L+S87P+A112P+T124V+D142L;

S5*+N26R+D79L+S87P+A112P+D142L;

N26R+T46R+D79L+S87P+A112P+D142L;

T46R+D79L+S87P+T116V+D142L;

D79L+P81R+S87P+A112P+D142L;

A27K+D79L+S87P+A112P+T124V+D142L;

D79L+Y82F+S87P+A112P+T124V+D142L;

D79L+Y82F+S87P+A112P+T124V+D142L;

D79L+S87P+A112P+T124V+A126V+D142L;

D79L+S87P+A112P+D142L;

D79L+Y82F+S87P+A112P+D142L;

S38T+D79L+S87P+A112P+A126V+D142L;

D79L+Y82F+S87P+A112P+A126V+D142L;

A27K+D79L+S87P+A112P+A126V+D142L;

D79L+S87P+N98C+A112P+G135C+D142L;

D79L+S87P+A112P+D142L+T141C+M161C;

S36P+D79L+S87P+A112P+D142L;

A37P+D79L+S87P+A112P+D142L;

S49P+D79L+S87P+A112P+D142L;

S50P+D79L+S87P+A112P+D142L;

D79L+S87P+D104P+A112P+D142L;

D79L+Y82F+S87G+A112P+D142L;

S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;

D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;

S70V+D79L+Y82F+S87G+A112P+D142L;

D79L+Y82F+S87G+D104P+A112P+D142L;

D79L+Y82F+S87G+A112P+A126V+D142L;

Y82F+S87G+S70V+D79L+D104P+A112P+D142L;

Y82F+S87G+D79L+D104P+A112P+A126V+D142L;

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;

A27K+Y82F+S87G+D104P+A112P+A126V+D142L:

A27K+D79L+Y82F+D104P+A112P+A126V+D142L;

A27K+Y82F+D104P+A112P+A126V+D142L;

A27K+D79L+S87P+A112P+D142L;

D79L+S87P+D142L.

In a preferred embodiment the thermostable protease is a variant of themature metallo protease disclosed as the mature part of SEQ ID NO: 2disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO2010/008841 or SEQ ID NO: 2 herein with the following mutations:

-   D79L+S87P+A112P+D142L;-   D79L+S87P+D142L; or-   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In an embodiment the protease variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQID NO: 2 herein.

The thermostable protease may also be derived from any bacterium as longas the protease has the thermostability properties defined according tothe invention.

In an embodiment the thermostable protease is derived from a strain ofthe bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfuprotease).

In an embodiment the protease is one shown as SEQ ID NO: 1 in U.S. Pat.No. 6,358,726-B1 (Takara Shuzo Company) and SEQ ID NO: 13 herein.

In an embodiment the thermostable protease is one disclosed in SEQ IDNO: 13 herein or a protease having at least 80% identity, such as atleast 85%, such as at least 90%, such as at least 95%, such as at least96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1 or SEQ ID NO: 13herein. The Pyroccus furiosus protease can be purchased from Takara Bio,Japan,

The Pyrococcus furiosus protease is a thermostable protease according tothe invention. The commercial product Pyrococcus furiosus protease (PfuS) was found (see Example 5) to have a thermostability of 110% (80°C./70° C.) and 103% (90° C./70° C.) at pH 4.5 determined as described inExample 2.

In one embodiment a thermostable protease has a thermostability value ofmore than 20% determined as Relative Activity at 80° C./70° C.determined as described in Example 2.

In an embodiment the protease has a thermostability of more than 30%,more than 40%, more than 50%, more than 60%, more than 70%, more than80%, more than 90%, more than 100%, such as more than 105%, such as morethan 110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70° C.

In an embodiment protease has a thermostability of between 20 and 50%,such as between 20 and 40%, such as 20 and 30% determined as RelativeActivity at 80° C./70° C.

In an embodiment the protease has a thermostability between 50 and 115%,such as between 50 and 70%, such as between 50 and 60%, such as between100 and 120%, such as between 105 and 115% determined as RelativeActivity at 80° C./70° C.

In an embodiment the protease has a thermostability value of more than10% determined as Relative Activity at 85° C./70° C. determined asdescribed in Example 2.

In an embodiment the protease has a thermostability of more than 10%,such as more than 12%, more than 14%, more than 16%, more than 18%, morethan 20%, more than 30%, more than 40%, more that 50%, more than 60%,more than 70%, more than 80%, more than 90%, more than 100%, more than110% determined as Relative Activity at 85° C./70° C.,

In an embodiment the protease has a thermostability of between 10 and50%, such as between 10 and 30%, such as between 10 and 25% determinedas Relative Activity at 85° C./70° C.

In an embodiment the protease has more than 20%, more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% determined as Remaining Activity at 80° C.; and/or

In an embodiment the protease has more than 20%, more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% determined as Remaining Activity at 84° C.

Determination of “Relative Activity” and “Remaining Activity” is done asdescribed in Example 2.

In an embodiment the protease may have a themostability for above 90,such as above 100 at 85° C. as determined using the Zein-BCA assay asdisclosed in Example 3.

In an embodiment the protease has a themostability above 60%, such asabove 90%, such as above 100%, such as above 110% at 85° C. asdetermined using the Zein-BCA assay.

In an embodiment protease has a themostability between 60-120, such asbetween 70-120%, such as between 80-120%, such as between 90-120%, suchas between 100-120%, such as 110-120% at 85° C. as determined using theZein-BCA assay.

In an embodiment the thermostable protease has at least 20%, such as atleast 30%, such as at least 40%, such as at least 50%, such as at least60%, such as at least 70%, such as at least 80%, such as at least 90%,such as at least 95%, such as at least 100% of the activity of theJTP196 protease variant or Protease Pfu determined by the AZCL-caseinassay described in the “Materials & Methods”-section.

Carbohydrate-Source Generating Enzyme Present and/or Added DuringLiquefaction

According to the invention an optional carbohydrate-source generatingenzyme, in particular a glucoamylase, preferably a thermostableglucoamylase, may be present and/or added in liquefaction together withan alpha-amylase and hemicellulase, preferably xylanase, having aMelting Point (DSC) above 80° C., and an optional endoglucanase having aMelting Point (DSC) above 70° C., and an optional a pullulanase and/oroptional phytase.

The term “carbohydrate-source generating enzyme” includes any enzymesgenerating fermentable sugars. A carbohydrate-source generating enzymeis capable of producing a carbohydrate that can be used as anenergy-source by the fermenting organism(s) in question, for instance,when used in a process of the invention for producing a fermentationproduct, such as ethanol. The generated carbohydrates may be converteddirectly or indirectly to the desired fermentation product, preferablyethanol. According to the invention a mixture of carbohydrate-sourcegenerating enzymes may be used, Specific examples include glucoamylase(being glucose generators), beta-amylase and maltogenic amylase (beingmaltose generators).

In a preferred embodiment the carbohydrate-source generating enzyme isthermostable. The carbohydrate-source generating enzyme, in particularthermostable glucoamylase, may be added together with or separately fromthe alpha-amylase and the thermostable protease.

In an embodiment the carbohydrate-source generating enzyme, preferably athermostable glucoamylase, has a Relative Activity heat stability at 85°C. of at least 20%, at least 30%, preferably at least 35% determined asdescribed in Example 4 (heat stability).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a relative activity pH optimum at pH 5.0 of at least90%, preferably at least 95%, preferably at least 97%, such as 100%determined as described in Example 4 (pH optimum).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a pH stability at pH 5.0 of at least at least 80%,at least 85%, at least 90% determined as described in Example 4 (pHstability).

In a specific and preferred embodiment the carbohydrate-sourcegenerating enzyme is a thermostable glucoamylase, preferably of fungalorigin, preferably a filamentous fungi, such as from a strain of thegenus Penicillium, especially a strain of Penicillium oxalicum, inparticular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in WO 2011/127802 (which is hereby incorporated by reference) andshown in SEQ ID NO: 14 herein.

In an embodiment the thermostable glucoamylase has at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99% or 100% identity to the mature polypeptide shown in SEQ ID NO:2 in WO 2011/127802 or SEQ ID NOs: 14 herein.

In an embodiment the carbohydrate-source generating enzyme, inparticular thermostable glucoamylase, is the Penicillium oxalicumglucoamylase shown in SEQ ID NO: 14 herein.

In a preferred embodiment the carbohydrate-source generating enzyme is avariant of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in WO 2011/127802 and shown in SEQ ID NO: 14 herein, having a K79Vsubstitution (referred to as “PE001”) (using the mature sequence shownin SEQ ID NO: 14 for numbering). The K79V glucoamylase variant hasreduced sensitivity to protease degradation relative to the parent asdisclosed in WO 2013/036526 (which is hereby incorporated by reference).

Contemplated Penicillium oxalicum glucoamylase variants are disclosed inWO 2013/053801 (which is hereby incorporated by reference).

In an embodiment these variants have reduced sensitivity to proteasedegradation.

In an embodiment these variant have improved thermostability compared tothe parent.

More specifically, in an embodiment the glucoamylase has a K79Vsubstitution (using SEQ ID NO: 14 herein for numbering), correspondingto the PE001 variant, and further comprises at least one of thefollowing substitutions or combination of substitutions:

-   T65A; Q327F; E501V; Y504T; Y504*; T65A+Q327F; T65A+E501V;    T65A+Y504T; T65A+Y504*; Q327F+E501V; Q327F+Y504T; Q327F+Y504*;    E501V+Y504T; E501V+Y504*; T65A+Q327F+E501V; T65A+Q327F+Y504T;    T65A+E501V+Y504T; Q327F+E501V+Y504T; T65A+Q327F+Y504*;    T65A+E501V+Y504*; Q327F+E501V+Y504*; T65A+Q327F+E501V+Y504T;    T65A+Q327F+E501V+Y504*; E501V+Y504T; T65A+K161S; T65A+Q405T;    T65A+Q327W; T65A+Q327F; T65A+Q327Y; P11F+T65A+Q327F;    R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; P2N+P4S+P11F+T65A+Q327F;    P11F+D26C+K33C+T65A+Q327F; P2N+P4S+P11F+T65A+Q327W+E501V+Y504T;    R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; P11F+T65A+Q327W;    P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; P11F+T65A+Q327W+E501V+Y504T;    T65A+Q327F+E501V+Y504T; T65A+S105P+Q327W; T65A+S105P+Q327F;    T65A+Q327W+S364P; T65A+Q327F+S364P; T65A+S103N+Q327F;    P2N+P4S+P11F+K34Y+T65A+Q327F; P2N+P4S+P11F+T65A+Q327F+D445N+V447S;    P2N+P4S+P11F+T65A+I172V+Q327F; P2N+P4S+P11F+T65A+Q327F+N502*;    P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E;    P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S;    P2N+P4S+P11F+T65A+Q327F+S377T; P2N+P4S+P11F+T65A+V325T+Q327W;    P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T;    P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T;    P2N+P4S+P11F+Q26N+K34Y+T65A+Q327F;    P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T;    P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;    P2N+P4S+T10D+T65A+Q327F+E501V+Y504T;    P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T;    K5A+P11F+T65A+Q327F+E501V+Y504T;    P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T;    P2N+T10E+E18N+T65A+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N;    P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A;    P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T;    P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T;    P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;    P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*;    P2N+P4S+P11F+T65A+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T;    K5A+P11F+T65A+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A;    P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+V79I+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; S255N+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+E74N+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T;    P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T;    P2N+P4S+P11F+165A+Q327F+S359N+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T;    P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or    P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

In a preferred embodiment the Penicillium oxalicum glucoamylase varianthas a K79V substitution using SEQ ID NO: 14 herein for numbering (PE001variant), and further comprises one of the following mutations:

-   P11F+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327F;-   P11F+D26C+K33C+T65A+Q327F;-   P2N+P4S+P11F+T65A+Q327W+E501V+Y504T;-   P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or-   P11F+T65A+Q327W+E501V+Y504T.

In an embodiment the glucoamylase variant, such as Penicillium oxalicumglucoamylase variant has at least 60%, such as at least 70%, such as atleast 75% identity, preferably at least 80%, more preferably at least85%, more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, but lessthan 100% identity to the mature polypeptide of SEQ ID NO: 14 herein.

The carbohydrate-source generating enzyme, in particular glycoamylase,may be added in amounts from 0.1-100 micrograms EP/g DS, such as 0.5-50micrograms EP/g DS, such as 1-25 micrograms EP/g DS, such as 2-12micrograms EP/g DS.

Pullulanase Present and/or Added During Liquefaction

Optionally a pullulanase may be present and/or added during liquefactionstep i) together with an alpha-amylase and a hemicellulase, preferablyxylanase, having a melting point (DSC) above 80° C. As mentioned above aprotease, a carbohydrate-source generating enzyme, preferably athermostable glucoamylase, may also optionally be present and/or addedduring liquefaction step i).

The pullulanase may be present and/or added during liquefaction step i)and/or saccharification step ii) or simultaneous saccharification andfermentation.

Pullulanases (E.C. 3.2.1.41, pullulan 6-glucano-hydrolase), aredebranching enzymes characterized by their ability to hydrolyze thealpha-1,6-glycosidic bonds in, for example, amylopectin and pullulan.

Contemplated pullulanases according to the present invention include thepullulanases from Bacillus amyloderamificans disclosed in U.S. Pat. No.4,560,651 (hereby incorporated by reference), the pullulanase disclosedas SEQ ID NO: 2 in WO 01/151620 (hereby incorporated by reference), theBacillus deramificans disclosed as SEQ ID NO: 4 in WO 01/151620 (herebyincorporated by reference), and the pullulanase from Bacillusacidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (herebyincorporated by reference) and also described in FEMS Mic. Let. (1994)115, 97-106.

Additional pullulanases contemplated according to the present inventionincluded the pullulanases from Pyrococcus woesei, specifically fromPyrococcus woesei DSM No. 3773 disclosed in WO 92/02614.

In an embodiment the pullulanase is a family GH57 pullulanase. In anembodiment the pullulanase includes an X47 domain as disclosed in WO2011/087836 (which are hereby incorporated by reference). Morespecifically the pullulanase may be derived from a strain of the genusThermococcus, including Thermococcus litoralis and Thermococcushydrothermalis, such as the Thermococcus hydrothermalis pullulanaseshown WO 2011/087836 truncated at the X4 site right after the X47domain. The pullulanase may also be a hybrid of the Thermococcuslitoralis and Thermococcus hydrothermalis pullulanases or a T.hydrothermalis/T. litoralis hybrid enzyme with truncation site X4disclosed in WO 2011/087836 (which is hereby incorporated by reference).

In another embodiment the pullulanase is one comprising an X46 domaindisclosed in WO 2011/076123 (Novozymes).

The pullulanase may according to the invention be added in an effectiveamount which include the preferred amount of about 0.0001-10 mg enzymeprotein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gramDS, more preferably 0.0001-0.010 mg enzyme protein per gram DS.Pullulanase activity may be determined as NPUN. An Assay fordetermination of NPUN is described in the “Materials & Methods”-sectionbelow.

Suitable commercially available pullulanase products include PROMOZYME400L, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (GenencorInt., USA), and AMANO 8 (Amano, Japan).

Phytase Present and/or Added During Liquefaction

Optionally a phytase may be present and/or added in liquefaction incombination with an alpha-amylase and hemicellulase, preferablyxylanase, having a melting point (DSC) above 80° C.

A phytase used according to the invention may be any enzyme capable ofeffecting the liberation of inorganic phosphate from phytic acid(myo-inositol hexakisphosphate) or from any salt thereof (phytates).Phytases can be classified according to their specificity in the initialhydrolysis step, viz. according to which phosphate-ester group ishydrolyzed first. The phytase to be used in the invention may have anyspecificity, e.g., be a 3-phytase (EC 3.1.3.8), a 6-phytase (EC3.1.3.26) or a 5-phytase (no EC number). In an embodiment the phytasehas a temperature optimum above 50° C., such as in the range from 50-90°C.

The phytase may be derived from plants or microorganisms, such asbacteria or fungi, e.g., yeast or filamentous fungi.

A plant phytase may be from wheat-bran, maize, soy bean or lily pollen.Suitable plant phytases are described in Thomlinson et al, Biochemistry,1 (1962), 166-171; Barrientos et al, Plant. Physiol., 106 (1994),1489-1495; WO 98/05785; WO 98/20139.

A bacterial phytase may be from genus Bacillus, Citrobacter, Hafnia,Pseudomonas, Buttiauxella or Escherichia, specifically the speciesBacillus subtilis, Citrobacter braakii, Citrobacter freundii, Hafniaalvei, Buttiauxella gaviniae, Buttiauxella agrestis, Buttiauxellanoackies and E. coli. Suitable bacterial phytases are described in Paverand Jagannathan, 1982, Journal of Bacteriology 151:1102-1108; Cosgrove,1970, Australian Journal of Biological Sciences 23:1207-1220; Greiner etal, Arch. Biochem. Biophys., 303, 107-113, 1993; WO 1997/33976; WO1997/48812, WO 1998/06856, WO 1998/028408; WO 2004/085638, WO2006/037327, WO 2006/038062, WO 2006/063588, WO 2008/092901, WO2008/116878; and WO 2010/034835.

A yeast phytase may be derived from genus Saccharomyces orSchwanniomyces, specifically species Saccharomyces cerevisiae orSchwanniomyces occidentails. The former enzyme has been described as aSuitable yeast phytases are described in Nayini et al, 1984,Lebensmittel Wissenschaft and Technologie 17:24-26; Wodzinski et al,Adv. Appl. Microbiol., 42, 263-303; AU-A-24840/95;

Phytases from filamentous fungi may be derived from the fungal phylum ofAscomycota (ascomycetes) or the phylum Basidiomycota, e.g., the genusAspergillus, Thermomyces (also called Humicola), Myceliophthora,Manascus, Penicillium, Peniophora, Agrocybe, Paxillus, or Trametes,specifically the species Aspergillus terreus, Aspergillus niger,Aspergillus niger var. awamori, Aspergillus ficuum, Aspergillusfumigatus, Aspergillus oryzae, T. lanuginosus (also known as H.lanuginosa), Myceliophthora thermophila, Peniophora lycii, Agrocybepediades, Manascus anka, Paxillus involtus, or Trametes pubescens.Suitable fungal phytases are described in Yamada et al., 1986, Agric.Biol. Chem. 322:1275-1282; Piddington et al., 1993, Gene 133:55-62; EP684,313; EP 0 420 358; EP 0 684 313; WO 1998/28408; WO 1998/28409; JP7-67635; WO 1998/44125; WO 1997/38096; WO 1998/13480.

In a preferred embodiment the phytase is derived from Buttiauxella, suchas Buttiauxella gaviniae, Buttiauxella agrestis, or Buttiauxellanoackies, such as the ones disclosed as SEQ ID NO: 2, SEQ ID NO: 4 andSEQ ID NO: 6, respectively, in WO 2008/092901 (hereby incorpotared byreference)

In a preferred embodiment the phytase is derived from Citrobacter, suchas Citrobacter braakii, such as one disclosed in WO 2006/037328 (herebyincorporated by reference).

Modified phytases or phytase variants are obtainable by methods known inthe art, in particular by the methods disclosed in EP 897010; EP 897985;WO 99/49022; WO 99/48330, WO 2003/066847, WO 2007/112739, WO2009/129489, and WO 2010/034835.

Commercially available phytase containing products include BIO-FEEDPHYTASE™, PHYTASE NOVO™ CT or L (all from Novozymes), LIQMAX (DuPont) orRONOZYME™ NP, RONOZYME® HiPhos, RONOZYME® P5000 (CT), NATUPHOS™ NG 5000(from DSM).

Carbohydrate-Source Generating Enzyme Present and/or Added DuringSaccharification and/or Fermentation

According to the invention a carbohydrate-source generating enzyme,preferably a glucoamylase, is present and/or added duringsaccharification and/or fermentation.

In a preferred embodiment the carbohydrate-source generating enzyme is aglucoamylase, of fungal origin, preferably from a stain of Aspergillus,preferably A. niger, A. awamori, or A. oryzae; or a strain ofTrichoderma, preferably T. reesei; or a strain of Talaromyces,preferably T. emersonii.

Glucoamylase

According to the invention the glucoamylase present and/or added insaccharification and/or fermentation may be derived from any suitablesource, e.g., derived from a microorganism or a plant. Preferredglucoamylases are of fungal or bacterial origin, selected from the groupconsisting of Aspergillus glucoamylases, in particular Aspergillus nigerG1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102),or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylasedisclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Agric. Biol.Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.Other Aspergillus glucoamylase variants include variants with enhancedthermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9,499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582);N182 (Chen et al. (1994), Biochem, J. 301, 275-281); disulphide bonds,A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; andintroduction of Pro residues in position A435 and S436 (Li et al.(1997), Protein Eng. 10, 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka et al. (1998) “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215). In a preferred embodiment theglucoamylase used during saccharification and/or fermentation is theTalaromyces emersonii glucoamylase disclosed in WO 99/28448.

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831),

Contemplated fungal glucoamylases include Trametes cingulata,Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed inWO 2006/069289; and Peniophora rufomarginata disclosed in WO2007/124285;or a mixture thereof. Also hybrid glucoamylase are contemplatedaccording to the invention. Examples include the hybrid glucoamylasesdisclosed in WO 2005/045018. Specific examples include the hybridglucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids arehereby incorporated by reference).

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus as described in WO2011/066576 (SEQ ID NOs 2, 4 or 6), or from a strain of the genusGloephyllum, in particular a strain of Gloephyllum as described in WO2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16) or a strain of thegenus Nigrofomes, in particular a strain of Nigrofomes sp, disclosed inWO 2012/064351 (SEQ ID NO: 2) (all references hereby incorporated byreference). Contemplated are also glucoamylases which exhibit a highidentity to any of the above-mentioned glucoamylases, i.e., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, such as100% identity to any one of the mature parts of the enzyme sequencesmentioned above.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably0.001-10 AGU/g DS, especially between 001-5 AGU/g DS, such as 0,1-2AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYME™ACHIEVE and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417(from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™G900, G-ZYME™ and G990 ZR (from Danisco US).

Maltogenic Amylase

The carbohydrate-source generating enzyme present and/or added duringsaccharification and/or fermentation may also be a maltogenicalpha-amylase. A “maltogenic alpha-amylase” (glucan1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amyloseand amylopectin to maltose in the alpha-configuration. A maltogenicamylase from Bacillus stearothermophilus strain NCIB 11837 iscommercially available from Novozymes A/S. Maltogenic alpha-amylases aredescribed in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, whichare hereby incorporated by reference. The maltogenic amylase may in apreferred embodiment be added in an amount of 0.05-5 mg totalprotein/gram DS or 0.05-5 MANU/g DS.

Cellulase or Cellulolytic Enzyme Composition Present and/or Added duringSaccharification and/or Fermentation or SSF

In a preferred embodiment of the invention a cellulase or cellulolyticenzyme composition is present and/or added in saccharification in stepii) and/or fermentation in step iii) or SSF.

The cellulase or cellulolytic enzyme composition may comprise one ormore cellulolytic enzymes. The cellulase or cellulolytic enzymecomposition may be of any origin. In a preferred embodiment thecellulase or cellulolytic enzyme composition comprises cellulolyticenzymes of fungal origin. In an embodiment the cellulase or cellulolyticenzyme composition is derived from a strain of Trichoderma, such asTrichoderma reesei; or a strain of Humicola, such as Humicola insolens:or a strain of Chrysosporium, such as Chrysosporium lucknowense; or astrain of Penicillium, such as Penicillium decumbens. In a preferredembodiment the cellulolytic enzyme composition is derived from a strainof Trichoderma reesei. The cellulase may be a beta-glucosidase, acellobiohydrolase, and an endoglucanase or a combination thereof. Thecellulolytic enzyme composition may comprise a beta-glucosidase, acellobiohydrolase, and an endoglucanase.

In an embodiment the cellulase or cellulolytic enzyme compositioncomprising one or more polypeptides selected from the group consistingof:

beta-glucosidase;

cellobiohydrolase I;

cellobiohydrolase II;

or a mixture thereof.

In a preferred embodiment the cellulase or cellulolytic enzymecomposition further comprises a GH61 polypeptide having cellulolyticenhancing activity. Cellulolytic enhancing activity is defined anddetermined as described in WO 2011/041397 (incorporated by reference).

The term “GH61 polypeptide having cellulolytic enhancing activity” meansa GH61 polypeptide that enhances the hydrolysis of a cellulosic materialby enzymes having cellulolytic activity. For purposes of the presentinvention, cellulolytic enhancing activity may be determined bymeasuring the increase in reducing sugars or the increase of the totalof cellobiose and glucose from hydrolysis of a cellulosic material bycellulolytic enzyme under the following conditions: 1-50 mg of totalprotein/g of cellulose in PCS (Pretreated Corn Stover), wherein totalprotein is comprised of 50-99.5% w/w cellulolytic enzyme protein and0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancingactivity for 1-7 days at 50° C. compared to a control hydrolysis withequal total protein loading without cellulolytic enhancing activity(1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferredaspect, a mixture of CELLUCLAST™ 1.5 L (Novozymes A/S, Bagsværd,Denmark) in the presence of 2-3% of total protein weight Aspergillusoryzae beta-glucosidase (recombinantly produced in Aspergillus oryzaeaccording to WO 02/095014) or 2-3% of total protein weight Aspergillusfumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzaeas described in WO 2002/095014) of cellulase protein loading is used asthe source of the cellulolytic activity.

The cellulolytic enzyme composition may comprise a beta-glucosidase,preferably one derived from a strain of the genus Aspergillus, such asAspergillus oryzae, such as the one disclosed in WO 2002/095014 or thefusion protein having beta-glucosidase activity disclosed in WO2008/057637 (see SEQ ID NOs: 74 or 76), or Aspergillus fumigatus, suchas one disclosed in SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 29herein; or an Aspergillus fumigatus beta-glucosidase variant disclosedin WO 2012/044915; or a strain of the genus a strain Penicillium, suchas a strain of the Penicillium brasilianum disclosed in WO 2007/019442,or a strain of the genus Trichoderma, such as a strain of Trichodermareesei.

In an embodiment the beta-glucosidase is from a strain of Aspergillus,such as a strain of Aspergillus fumigatus, such as Aspergillus fumigatusbeta-glucosidase (SEQ ID NO: 29 herein), or a variant thereof, whichvariant comprises one or more substitutions selected from the groupconsisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y;such as a variant thereof with the following substitutions:

F100D+S283G+N456E+F512Y;

L89M+G91L+I186V+I140V;

I186V+L89M+I140V+F100D+S283G+N456E+F512Y (using SEQ ID NO: 29 herein fornumbering).

The parent beta-glucosidase may have at least 60% identity, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95%, such as at least 96%, such as at least 97%, such as at least 98%,such as at least 99%, such as 100% identity to the mature polypeptide ofSEQ ID NO: 29 herein.

In case the beta-glucosidase is a beta-glucosidase variant it may haveat least 60% identity, such as at least 70%, such as at least 80%, suchas at least 90%, such as at least 95%, such as at least 96%, such as atleast 97%, such as at least 98%, such as at least 99%, but less than100% identity to the mature polypeptide of SEQ ID NO: 29 herein.

In case the cellulolytic enzyme composition may comprise a GH61polypeptide, it may be one derived from the genus Thermoascus, such as astrain of Thermoascus aurantiacus, such as the one described in WO2005/074656 as SEQ ID NO: 2 or SEQ ID NO: 30 herein; or one derived fromthe genus Thielavia, such as a strain of Thielavia terrestris, such asthe one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; orone derived from a strain of Aspergillus, such as a strain ofAspergillus fumigatus, such as the one described in WO 2010/138754 asSEQ ID NO: 1 and SEQ ID NO: 2; or one derived from a strain derived fromPenicillium, such as a strain of Penicillium emersonii, such as the onedisclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 31 herein.

In a preferred embodiment the GH61 polypeptide, such as one derived froma strain of Penicillium, preferably a strain of Penicillium emersonii,is selected from the group consisting of:

-   (i) a GH61 polypeptide comprising the mature polypeptide of SEQ ID    NO: 31 herein;-   (ii) a GH61 polypeptide comprising an amino acid sequence having at    least 60%, such as at least 70%, e.g., at least 75%, at least 80%,    at least 85%, at least 90%, at least 91%, at least 92%, at least    93%, at least 94%, at least 95%, at least 96%, at least 97%, at    least 98%, or at least 99% identity to the mature polypeptide of SEQ    ID NO: 31 herein.

In an embodiment the cellulolytic enzyme composition comprises acellobiohydrolase I (CBH I), such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus, such asthe Cel7a CBH I disclosed in SEQ ID NO: 6 in WO 2011/057140 or SEQ IDNO: 32 herein, or a strain of the genus Trichoderma, such as a strain ofTrichoderma reesei.

In a preferred embodiment the cellobiohydrolase I, such as one derivedfrom a strain of Aspergillus, preferably a strain of Aspergillusfumigatus, is selected from the group consisting of:

-   (i) a cellobiohydrolase I comprising the mature polypeptide of SEQ    ID NO: 32 herein;-   (ii) a cellobiohydrolase I comprising an amino acid sequence having    at least 60%, such as at least 70%, e.g., at least 75%, at least    80%, at least 85%, at least 90%, at least 91%, at least 92%, at    least 93%, at least 94%, at least 95%, at least 96%, at least 97%,    at least 98%, or at least 99% identity to the mature polypeptide of    SEQ ID NO: 32 herein.

In an embodiment the cellulolytic enzyme composition comprises acellobiohydrolase II (CBH II), such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus; such asthe one disclosed as SEQ ID NO: 33 herein or a strain of the genusTrichoderma, such as Trichoderma reesei, or a strain of the genusThielavia, such as a strain of Thielavia terrestris, such ascellobiohydrolase II CEL6A from Thielavia terrestris.

In a preferred embodiment cellobiohydrolase II, such as one derived froma strain of Aspergillus, preferably a strain of Aspergillus fumigatus,is selected from the group consisting of:

-   (i) a cellobiohydrolase II comprising the mature polypeptide of SEQ    ID NO: 33 herein.-   (ii) a cellobiohydrolase II comprising an amino acid sequence having    at least 60%, such as at least 70%, e.g., at least 75%, at least    80%, at least 85%, at least 90%, at least 91%, at least 92%, at    least 93%, at least 94%, at least 95%, at least 96%, at least 97%,    at least 98%, or at least 99% identity to the mature polypeptide of    SEQ ID NO: 33 herein.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity and abeta-glucosidase.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity derived from a strainof Penicillium, such as a strain of Penicillium emersonii, such as theone disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 31 herein,and a beta-glucosidase.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,and a CBH I.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity derived from a strainof Penicillium, such as a strain of Penicillium emersonii, such as theone disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 31 herein,a beta-glucosidase, and a CBHI.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,a CBHI, and a CBHII.

In an embodiment the cellulolytic enzyme composition comprises a GH61polypeptide having cellulolytic enhancing activity derived from a strainof Penicillium, such as a strain of Penicillium emersonii, such as theone disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 31 herein,a beta-glucosidase, a CBHI, and a CBHII.

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide (SEQ ID NO: 2 in WO 2005/074656 or SEQ IDNO: 30 herein), and Aspergillus oryzae beta-glucosidase fusion protein(WO 2008/057637).

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity(SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 30 herein) and Aspergillusfumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO:29 herein).

In an embodiment the cellulolytic enzyme composition is a Trichodermareesei cellulolytic enzyme composition further comprising Penicilliumemersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397or SEQ ID NO: 31 herein, and Aspergillus fumigatus beta-glucosidasedisclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 29 herein, ora variant thereof, which variant has one of, preferably all of, thefollowing substitutions: F100D, S283G, N456E, F512Y.

In an embodiment the cellulolytic enzyme composition comprises one ormore of the following components:

-   (i) an Aspergillus fumigatus cellobiohydrolase I;-   (ii) an Aspergillus fumigatus cellobiohydrolase II;-   (iii) an Aspergillus fumigatus beta-glucosidase or variant thereof.

In an embodiment the Aspergillus fumigatus beta-glucosidase (SEQ ID NO:29 herein), comprises one or more substitutions selected from the groupconsisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y;such as a variant thereof, with the following substitutions:

-   F100D+S283G+N456E+F512Y;-   L89M+G91L+I186V+I140V; or-   I186V+L89M+G91L+I140V+F100D+S283G+N456E+F512Y.

In an embodiment the cellulolytic enzyme composition further comprisesthe Penicillium sp. GH61 polypeptide shown in SEQ ID NO: 31 herein; or aGH61 polypeptide comprising an amino acid sequence having at least 60%,such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,such as 100% identity to the mature polypeptide of SEQ ID NO: 31 herein.

In an embodiment the cellulolytic enzyme composition comprising thefollowing components:

-   (i) Aspergillus fumigatus cellobiohydrolase I shown in SEQ ID NO: 32    herein;-   (ii) Aspergillus fumigatus cellobiohydrolase II shown in SEQ ID NO:    33 herein;-   (iii) a variant of Aspergillus fumigatus beta-glucosidase shown in    SEQ ID NO: 29 with the following substitutions:    F100D+S283G+N456E+F512Y; and-   (iv) Penicillium sp. GH61 polypeptide shown in SEQ ID NO: 31 herein.

In an embodiment cellulolytic enzyme composition is dosed (i.e, duringsaccharification in step ii) and/or fermentation in step iii) or SSF)from 0.0001-3 mg EP/g DS, preferably 0.0005-2 mg EP/g DS, preferably0.001-1 mg/g DS, more preferred from 0.005-0.5 mg EP/g DS, even morepreferred 0.01-0.1 mg EP/g DS.

Examples of Preferred Processes of the Invention

In a preferred embodiment the invention relates to a process forproducing fermentation products from starch-containing materialcomprising the steps of:

i) liquefying the starch-containing material at a pH in the rangebetween from above 4.0-6.5 at a temperature in the range from 70-100° C.using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C.    -   an optional endoglucanase having a Melting Point (DSC) above 70°        C.;

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism.

In a preferred embodiment the process of the invention comprises thesteps of:

-   i) liquefying the starch-containing material at a pH in the range    between from above 4.5-6.2 at a temperature above the initial    gelatinization temperature using:    -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C.;    -   an optional endoglucanase having a Melting Point (DSC) above 70°        C.;-   ii) saccharifying using a glucoamylase enzyme;-   iii) fermenting using a fermenting organism.

In a preferred embodiment the process of the invention comprises thesteps of:

-   i) liquefying the starch-containing material at a pH in the range    between from above 4.0-6.5 at a temperature between 70-100° C.:    -   a bacterial alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C;—an optional endoglucanase having a Melting        Point (DSC), between 70° C. and 95° C.;    -   optionally a protease, preferably derived from Pyrococcus        furiosus or Thermoascus aurantiacus, having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.;-   ii) saccharifying using a glucoamylase enzyme;-   iii) fermenting using a fermenting organism.

In a preferred embodiment the process of the invention comprises thesteps of:

-   i) liquefying the starch-containing material at a pH in the range    between from above 4.0-6.5 at a temperature above the initial    gelatinization temperature using:    -   an alpha-amylase shown in SEQ ID NO: 1 having a double deletion        in positions R179+G180 or I181+G182, and optional substitution        N193F; and optionally further one of the following set of        substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q2545S+M284V (using SEQ ID NO: 1        herein for numbering);    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C.; such as an xylanase having at least 60%,        such as at least 70%, such as at least 75% identity, preferably        at least 80%, more preferably at least 85%, more preferably at        least 90%, more preferably at least 91%, more preferably at        least 92%, even more preferably at least 93%, most preferably at        least 94%, and even most preferably at least 95%, such as even        at least 96%, at least 97%, at least 98%, at least 99%, such as        100% identity to the mature part of the polypeptide of SEQ ID        NOs: 3, 4, 5, 8 and 34 herein; preferably SEQ ID NO: 5 herein;    -   an optional endoglucanase having a Melting Point (DSC), between        70° C. and 95° C.; such as an endoglucanase having at least 60%,        such as at least 70%, such as at least 75% identity, preferably        at least 80%, more preferably at least 85%, more preferably at        least 90%, more preferably at least 91%, more preferably at        least 92%, even more preferably at least 93%, most preferably at        least 94%, and even most preferably at least 95%, such as even        at least 96%, at least 97%, at least 98%, at least 99%, such as        100% to the mature part of the any of the polypeptides shown in        SEQ ID NOs: 9, 35, 36, 37 or 38 herein;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.        derived from Pyrococcus furiosus and/or Thermoascus aurantiacus;    -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 14        herein, preferably having substitutions selected from the group        of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; (using SEQ ID NO: 14 for        numbering);    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism.

In a preferred embodiment the process of the invention comprises thesteps of:

-   i) liquefying the starch-containing material at a pH in the range    between from above 4.0-6.5 at a temperature between 70-100° C.    using:    -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion in positions I181+G182, and optional        substitution N193F; and optionally further one of the following        set of substitutions:    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S: or    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1        herein (using SEQ ID NO: 1 herein for numbering);    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C.;    -   such as an xylanase having at least 60%, such as at least 70%,        such as at least 75% identity, preferably at least 80%, more        preferably at least 85%, more preferably at least 90%, more        preferably at least 91%, more preferably at least 92%, even more        preferably at least 93%, most preferably at least 94%, and even        most preferably at least 95%, such as even at least 96%, at        least 97%, at least 98%, at least 99%, such as 100% identity to        the mature part of the polypeptide of SEQ ID NOs: 3, 4, 5, 8 and        34 herein; preferably SEQ ID NO: 5 herein    -   an optional endoglucanase having a Melting Point (DSC), between        70° C. and 95° C.; preferably having at least 90% identity to        the mature part of the polypeptide of SEQ ID NO: 9 herein;    -   a optional protease having a thermostability value of more than        20% determined as Relative Activity at 80° C./70° C. derived        from Pyrococcus furiosus and/or Thermoascus aurantiacus; and    -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 14        herein, preferably having substitutions selected from the group        of:    -   K79V; or    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 herein for        numbering);    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism.

In a preferred embodiment the process of the invention comprises thesteps of:

-   i) liquefying the starch-containing material at a pH in the range    between from above 4.0-6.5 at a temperature between 70-100° C.    using:    -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion in positions I181+G182, and optional        substitution N193F; and optionally further one of the following        set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering);    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C.;    -   an optional endoglucanase having a Melting Point (DSC), between        70° C. and 95° C.; preferably having at least 90% identity to        the mature part of the polypeptide of SEQ ID NO: 9 herein;    -   a protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C. derived from        Pyrococcus furiosus;    -   a Penicillium oxalicum glucoamylase in SEQ ID NO: 14 herein,        preferably having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 herein for        numbering);-   ii) saccharifying using a glucoamylase enzyme;-   iii) fermenting using a fermenting organism.

In a preferred embodiment a cellulase or cellulolytic enzyme compositionis present and/or added during fermentation or simultaneoussaccharification and fermentation.

In a preferred embodiment a cellulase or cellulolytic enzyme compositionderived from Trichoderma reesei is present and/or added duringfermentation or simultaneous saccharification and fermentation (SSF).

In a preferred embodiment a cellulase or cellulolytic enzyme compositionand a glucoamylase are present and/or added during fermentation orsimultaneous saccharification and fermentation.

In an embodiment the cellulase or cellulolytic enzyme composition isderived from Trichoderma reesei, Humicola insolens, Chrysosporiumlucknowense or Penicillium decumbens.

A Composition of the Invention

A composition of the invention comprises an alpha-amylase, such as athermostable alpha-amylase, and a hemicellulase, preferably xylanase,having a Melting Point (DSC) above 80° C.; an optional endoglucanasehaving a Melting Point (DSC) above 70° C.; an optional protease, such asa thermostable protease. The composition may also further comprise acarbohydrate-source generating enzyme, in particular a glucoamylase,optionally a pullulanase and optionally a phytase too.

Therefore, in this aspect the invention relates to compositioncomprising:

-   -   an alpha-amylase;    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C.    -   an optional endoglucanase having a Melting Point (DSC) above 70°        C.;    -   optionally a protease;    -   optionally a carbohydrate-source generating enzyme.

Alpha-amylase: The alpha-amylase may be any alpha-amylase. In apreferred embodiment the alpha-amylase is a bacterial alpha-amylases,such as alpha-amylases derived from the genus Bacillus, such as Bacillusstearomthermphilus, preferably the one shown in SEQ ID NO: 1 herein.

The alpha-amylase may be a thermostable alpha-amylase. The thermostablealpha-amylase may have a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) ofat least 10, such as at least 15, such as at least 20, such as at least25, such as at least 30, such as at least 40, such as at least 50, suchas at least 60, such as between 10-70, such as between 15-70, such asbetween 20-70, such as between 25-70, such as between 30-70, such asbetween 40-70, such as between 50-70, such as between 60-70.

In an embodiment the alpha-amylase is selected from the group ofBacillus stearomthermphilus alpha-amylase variants, in particulartruncated to be 491 amino acids long, such as from 480 to 495 aminoacids long, with mutations selected from the group of:

-   I181*+G182*;-   I181*+G182*+N193F;-   preferably-   I181*+G182*+E129V+K177L+R179E;-   I181*+G182*+N193F+E129V+K177L+R179E;-   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;-   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and-   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using    SEQ ID NO: 1 herein for numbering).

It should be understood that these alpha-amylases are only specificexamples. Any alpha-amylase disclosed above in the “Alpha-AmylasePresent and/or Added During Liquefaction”-section above may be used asthe alpha-amylase component in a composition of the invention.

Endocilucanase: According to the invention an optional endoglucanasecomponent may be comprised in the composition. It may be anyendoglucanase having a Melting Point (DSC) above 70° C., such as above75° C., in particular above 80° C., such as between 70° C. and 95° C.,determined using the “Differential Scanning calorimetry (DSC) Assay”described in the “Materials & Methods”—section below.

In an embodiment the endoglucanase has a Melting Point (DSC) above 72°C., such as above 74° C., such as above 76° C., such as above 78° C.,such as above 80° C., such as above 82° C. such as above 84° C., such asabove 86° C., such as above 88° C., such as between 70° C. and 95° C.,such as between 76° C. and 94° C., such as between 78° C. and 93° C.,such as between 80° C. and 92° C., such as between 82° C. and 91° C.,such as between 84° C. and 90° C.

In a preferred embodiment the endoglucanase used in a process of theinvention comprised in a composition of the invention is a GlycosideHydrolase Family 5 endoglucnase or GH5 endoglucanase (see the CAZydatabase on the “www.cazy.org” webpage. In an embodiment the GH5endoglocianase is from family EG II, such as the Talaromyces leycettanusendoglucanase shown in SEQ ID NO: 9 herein; Penicillium capsulatumendoglucanase show in SEQ ID NO: 35 herein, and Trichophaea saccataendoglucanase shown in SEQ ID NO: 36 herein.

In an embodiment the endoglucanase is a family GH45 endoglucanase. In anembodiment the GH45 endoglocianase is from family EG V, such as theSordana fimicola shown in SEQ ID NO: 38 herein or Thielavia terrestrisendoglucnase shown in SEQ ID NO: 37 herein.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 9 herein. In an embodiment the endoglucanase is derivedfrom a strain of the genus Talaromyces, such as a strain of Talaromycesleycettanus.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 35 herein, preferably derived from a strain of the genusPenicillium, such as a strain of Penicillium capsulatum.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 36 herein, preferably derived from a strain of the genusTrichophaea, such as a strain of Trichophaea saccata.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 37 herein, preferably derived from a strain of the genusThielavia, such as a strain of Thielavia terrestris.

In an embodiment the endoglucanase has at least 60%, such as at least70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NO: 38 herein, preferably derived from a strain of the genusSordaria, such as a strain of Sordaria fimicola.

It should be understood that these endoglucanases are only specificexamples. Any endoglucanase disclosed above in the “ThermostableEndoglucanase Present and/or Added During Liquefaction”-section abovemay be used as the optional endoglucoanase component in a composition ofthe invention.

In an especially preferred embodiment the endoglucanase (EG) has atleast 90% identity to the mature part of the polypeptide of SEQ ID NO: 9herein derived from a strain of Talaromyces leycettanus having a MeltingPoint (DSC) above 80° C.

Protease: A composition of the invention may optionally comprise aprotease, such as a thermosyable protease. There is no limitation on theorigin of the protease component as long as it fulfills thethermostability properties defined herein.

In an embodiment the protease is of fungal origin. In an embodiment theprotease is a metallo protease. In an embodiment the protease is derivedfrom Thermoascus aurantiacus shown in SEQ ID NO: 2 herein.

In a preferred embodiment the protease is a variant of the Thermoascusaurantiacus protease mentioned above having a thermostability value ofmore than 20% determined as Relative Activity at 80° C./70° C.determined as described in Example 2.

In a specific preferred embodiment the protease is a variant of themetallo protease derived from Thermoascus aurantiacus disclosed as themature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the maturepart of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 2 herein withmutations selected from the group of:

-   -   D79L+S87P+A112P+D142L;    -   D79L+S87P+D142L; and    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In another embodiment the protease is a bacterial protease. In anotherembodiment the protease is a serine protease. In a preferred embodimentthe protease is derived from a strain of Pyrococcus furiosus, such asthe one shown in SEQ ID NO: 1 in U.S. Pat. No. 6,358,726 or SEQ ID NO:13 herein.

It should be understood that these proteases are only examples. Anyprotease disclosed above in the “Protease Present and/or Added DuringLiquefaction” section above may be used as the protease component in acomposition of the invention.

Carbohydrate-source generating enzymes: A composition of the inventionmay optionally further comprise a carbohydrate-source generating enzyme,in particular a glucoamylase, such as a thermostable glucoamylase whichhas a heat stability at 85° C., pH 5.3, of at least 30%, preferably atleast 35%,

Said carbohydrate-source generating enzyme may be a thermostableglucoamylase having a Relative Activity heat stability at 85° C. of atleast 20%, at least 30%, preferably at least 35% determined as describedin Example 4 (Heat stability).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a relative activity pH optimum at pH 5.0 of at least90%, preferably at least 95%, preferably at least 97%, such as 100%determined as described in Example 4 (pH optimum).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a pH stability at pH 5.0 of at least at least 80%,at least 85%, at least 90% determined as described in Example 4 (pHstability).

In a preferred embodiment the carbohydrate-source generating enzyme is athermostable glucoamylase, preferably of fungal origin, preferably afilamentous fungi, such as from a strain of the genus Penicillium,especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 inWO 2011/127802 (which is hereby incorporated by reference), or a variantthereof, and shown in SEQ ID NO: 14 herein.

In an embodiment the glucoamylase, or a variant thereof, may have atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, such as 100% identity to the mature polypeptideshown in SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NO: 14 herein.

In a specific and preferred embodiment the carbohydrate-sourcegenerating enzyme is a variant of the Penicillium oxalicum glucoamylasedisclosed as SEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 14herein, having a K79V substitution (using the mature sequence shown inSEQ ID NO: 14 herein for numbering). The K79V glucoamylase variant hasreduced sensitivity to protease degradation relative to the parent asdisclosed in WO 2013/036526 (which is hereby incorporated by reference).

Examples of suitable thermostable Penicillium oxalicum glucoamylasevariants are listed above and in Examples 17 and 18 below.

In an embodiment the carbohydrate-source generating enzyme, such asglucoamyase, such as Penicillium oxalicum glucoamylase, has pullulanaseside-activity.

It should be understood that these carbohydrate-source generatingenzymes, in particular glucoamylases, are only examples. Anycarbohydrate-source generating enzyme disclosed above in the“Carbohydrate-source generating enzyme Present and/or Added DuringLiquefaction” section above may be used as component in a composition ofthe invention.

In a preferred embodiment the the carbohydrate-source generating enzymeis the Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14 hereinor a sequence having at least 90% identity thereto further comprising aK79V substitution.

Pullulanase: A composition of the invention may optionally furthercomprise a pullulanase. The pullulanase may be of any origin.

In an embodiment the pullulanase is of bacterial origin. In anembodiment the pullulanase is derived from a strain of Bacillus sp.

In an embodiment the pullulanase is a family GH57 pullulanase. In apreferred embodiment the pullulanase includes an X47 domain as disclosedin WO 2011/087836 (which is hereby incorporated by reference).

Specifically the pullulanase may be derived from a strain from the genusThermococcus, including Thermococcus litoralis and Thermococcushydrothermalis or a hybrid thereof.

The pullulanase may be Thermococcus hydrothermalis pullulanase shown inSEQ ID NO: 11 herein truncated at site X4 or a Thermococcushydrothermalis/T. litoralis hybrid enzyme (SEQ ID NO: 12 herein) withtruncation site X4 as disclosed in WO 2011/087836.

The another embodiment the pullulanase is one comprising an X46 domaindisclosed in WO 2011/076123 (Novozymes).

It should be understood that these pullulanases are only specificexamples. Any pullulanase disclosed above in the “Pullulanase Presentand/or Added During Liquefaction”-section above may be used as theoptional pullulanase component in a composition of the invention.

Phytase: A composition of the invention may optionally further comprisea phytase. The phytase may be of any origin.

In an embodiment the phytase is of bacterial origin. In an embodimentthe phytase is derived from a strain of from Buttiauxella, such asButtiauxella gaviniae, such as the one disclosed as SEQ ID NO: 2 (aminoacids 1-33 are expected signal peptide) in WO 2008/092901; orButtiauxella agrestis, such as the one shown as SEQ ID NO: 4 (aminoacids −9 to −1 are expected to be a part of the signal peptide) in WO2008/092901; or Buttiauxella noackies, such as the one shown as SEQ IDNO: 6 in WO 2008/092901.

In another embodiment the phytase is derived from a strain ofCitrobacter, such as a strain of Citrobacter braakii, such as onesdisclosed as SEQ ID NOs: 2 or 4 in WO 2006/037328 (hereby incorporatedby reference).

It should be understood that these phytases are only specific examples.Any phytase disclosed above in the “Phytase Present and/or Added DuringLiquefaction”-section above may be used as the optional pullulanasecomponent in a composition of the invention.

In a preferred embodiment the phytase is derived from a strain ofButtiauxella.

Examples of Preferred Embodiments of the Composition of the Invention

In a preferred embodiment the composition of the invention comprises

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   a hemicellulase, preferably xylanase, having a Melting Point        (DSC) above 80° C.;    -   an optional endoglucanase having a Melting Point (DSC) above 70°        C., such as between 70° C. and 95° C.;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.        derived from Pyrococcus furiosus or Thermoascus auranticus; and    -   optionally a glucoamylase, such as one derived from Penicillium        oxalicum.

In another embodiment the composition of the invention comprises

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus; having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   a hemicellulase, preferably a xylanase, having a Melting Point        (DSC) above 80° C.;    -   an optional endoglucanase having a Melting Point (DSC) between        70° C. and 95° C.;        -   an optional protease, preferably derived from Pyrococcus            furiosus or Thermoascus aurantiacus, having a            thermostability value of more than 20% determined as            Relative Activity at 80° C./70° C.; and        -   optionally a glucoamylase, e.g., derived from Penicillium            oxalicum.

In another embodiment the composition of the invention comprises

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion in positions I181+G182, and optionally        substitution N193F; and optionally further one of the following        set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering);    -   a hemicellulase having a Melting Point (DSC) above 80° C.;    -   an optional endoglucanase having a Melting Point (DSC) above 70°        C.;    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus, having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C.; and    -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 14        having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T, or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T, or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase (SEQ IDNO: 1 herein), or a variant thereof, is the mature alpha-amylase orcorresponding mature alpha-amylases having at least 80% identity, atleast 90% identity, at least 95% identity at least 96% identity at least97% identity at least 99% identity to the SEQ ID NO: 1 herein.

In an embodiment the hemicellulase, in particular xylanase, especiallyGH10 or GH11 xylanase has a Melting Point (DSC) above 82° C., such asabove 84° C., such as above 86° C., such as above 88° C., such as above88° C., such as above 90° C., such as above 92° C., such as above 94°C., such as above 96° C., such as above 98° C., such as above 100° C.,such as between 80° C. and 110° C., such as between 82° C. and 110° C.,such as between 84° C. and 110° C.

Examples of suitable hemicellulases, in particular xylanases, includethe xylanase shown in SEQ ID NOs: 3 herein or SEQ ID NO: 34 hereinderived from a strain of Dictyogllomus thermophilum; the xylanase shownin SEQ ID NO: 4 herein derived from a strain of Rasomsoniabyssochlamydoides; the xylanase shown in SEQ ID NO: 5 herein derivedfrom a strain of Talaromyces leycettanus; the xylanase shown in SEQ IDNO: 8 herein derived from a strain of Aspergillus fumigatus or apolypeptide having hemicellulase, preferably xylanase activity, havingat least 60%, such as at least 70%, such as at least 75% identity,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, such as 100% identity to themature part of the polypeptides of SEQ ID NOs: 3, 4, 5, 8 and 34 herein.

In an embodiment the optional endoglucoanase has a Melting Point (DSC)above 74° C., such as above 76° C., such as above 78° C., such as above80° C., such as above 82° C., such as above 84° C., such as above 86°C., such as above 88° C., such as between 70° C. and 95° C., such asbetween 76° C. and 94° C., such as between 78° C. and 93° C., such asbetween 80° C. and 92° C., such as between 82° C. and 91° C., such asbetween 84° C. and 90° C.

In an embodiment the optional endoglucanase has at least 60%, such as atleast 70%, such as at least 75% identity, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, such as 100% identity to the mature part of the polypeptideof SEQ ID NOs: 9, 35, 36, 37 or 38 herein.

In an embodiment the endoglucanase has at least 80% identity to themature part of the polypeptide of SEQ ID NO: 9 herein.

In an embodiment the endoglucanase has at least 90% identity to themature part of the polypeptide of SEQ ID NO: 9 herein having a MeltingPoint (DSC) above 70° C.

In an embodiment the Pyrococcus furiosus protease (SEQ ID NO: 13 herein)and/or Thermoascus aurantiacus protease (SEQ ID NO: 2 herein), or avariant thereof, is the mature protease or corresponding mature proteasehaving at least 80% identity, at least 90% identity, at least 95%identity at least 96% identity at least 97% identity at least 99%identity to the SEQ ID NO: 2 herein or SEQ ID NO: 13 herein,respectively,

In an embodiment 1 the Penicillium oxalicum glucoamylase (SEQ ID NO: 14herein), or a variant thereof, is the mature glucoamylase orcorresponding mature glucoamylase having at least 80% identity, at least90% identity, at least 95% identity at least 96% identity at least 97%identity at least 99% identity to the SEQ ID NO: 14 herein.

Further Aspects of the Invention

In a further aspect of the invention it relates to the use of acomposition of the invention for liquefying a starch-containingmaterial.

In a final aspect of the invention is relates to methods of producingliquefied starch, comprising liquefying a starch-containing materialwith a composition of the invention.

The invention is further summarized in the following paragraphs:

1. A process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase;    -   a hemicellulase having a Melting Point (DSC) above 80° C.;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism.

2. The process of paragraph 1, wherein the hemicellulase is a xylanase,in particular a GH10 xylanase or a GH11 xylanase.

3. The process of paragraph 1 or 2, wherein the hemicellulase, inparticular xylanase, especially GH10 or GH11 xylanase has a MeltingPoint (DSC) above 82° C., such as above 84° C., such as above 86° C.,such as above 88° C., such as above 88° C., such as above 90° C., suchas above 92° C., such as above 94° C., such as above 96° C., such asabove 98° C., such as above 100° C., such as between 80° C. and 110° C.,such as between 82° C. and 110° C., such as between 84° C. and 110° C.

4. The process of any of paragraphs 1-3, wherein the hemicellulase, inparticular xylanase, especially GH10 or GH11 xylanase has at least 60%,such as at least 70%, such as at least 75%, preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, such as 100% identity to the mature part of thepolypeptide of SEQ ID NO: 3 herein (GH10) or SEQ ID NO: 34 herein(GH11), preferably derived from a strain of the genus Dictyoglomus, suchas a strain of Dictyogilomus thermophilum.

5. The process any of paragraphs 1-3, wherein the hemicellulase, inparticular xylanase, especially GH10 xylanase has at least 60%, such asat least 70%, such as at least 75% identity, preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, such as 100% identity to the mature part of thepolypeptide of SEQ ID NO: 4 herein, preferably derived from a strain ofthe genus Rasamsonia, such as a strain of Rasomsonia byssochlamydoides.

6. The process of any of paragraphs 1-3, wherein the hemicellulase, inparticular xylanase, especially GH10 xylanase has at least 60%, such asat least 70%, such as at least 75% identity, preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, such as 100% identity to the mature part of thepolypeptide of SEQ ID NO: 5 herein, preferably derived from a strain ofthe genus Talaromyces, such as a strain of Talaromyces leycettanus.

7. The process of paragraphs 1 or 2, wherein the hemicellulase, inparticular xylanase, especially GH10 xylanase has at least 60%, such asat least 70%, such as at least 75% identity, preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, such as 100% identity to the mature part of thepolypeptide of SEQ ID NO: 8 herein, preferably derived from a strain ofthe genus Aspergillus, such as a strain of Aspergillus fumigatus.

8. The process of any of paragraphs 1-7, wherein

an alpha-amylase;

a hemicellulase having a Melting Point (DSC) above 80° C., preferablyabove 85° C., especially above 90° C., in particular 95° C.;

an optional endoglucanase having Melting Point (DSC) above 70° C.,preferably above 75° C., in particular above 80° C., especially above85° C.; are present and/or added in liquefaction step i).

9. The process of any of paragraphs 1-8, further comprises, prior to theliquefaction step i), the steps of:

-   -   a) reducing the particle size of the starch-containing        material_(;)preferably by dry milling;    -   b) forming a slurry comprising the starch-containing material        and water.

10. The process of any of paragraphs 1-9, wherein at least 50%,preferably at least 70%, more preferably at least 80%, especially atleast 90% of the starch-containing material fit through a sieve with #6screen.

11. The process of any of paragraphs 1-10, wherein the pH duringliquefaction is between 4.0-6.5, such as 4.5-6.2, such as above 4.8-6.0,such as between 5.0-5.8.

12. The process of any of paragraphs 1-11, wherein the temperatureduring liquefaction is in the range from 70-100° C., such as between70-95° C., such as between 75-90° C., preferably between 80-90° C., suchas around 85° C.

13. The process of any of paragraphs 1-12, wherein a jet-cooking step iscarried out before liquefaction in step i).

14. The process of paragraph 13, wherein the jet-cooking is carried outat a temperature between 95-160° C., such as between 110-145° C.,preferably 120-140° C., such as 125-135° C., preferably around 130° C.for about 1-15 minutes, preferably for about 3-10 minutes, especiallyaround about 5 minutes.

15. The process of any of paragraphs 1-14, wherein saccharification andfermentation is carried out sequentially or simultaneously.

16. The process of any of paragraphs 1-15, wherein saccharification iscarried out at a temperature from 20-75° C., preferably from 40-70° C.,such as around 60° C., and at a pH between 4 and 5.

17. The process of any of paragraphs 1-16, wherein fermentation orsimultaneous saccharification and fermentation (SSF) is carried out at atemperature from 25° C. to 40° C., such as from 28° C. to 35° C., suchas from 30° C. to 34° C., preferably around about 32° C., such as for 6to 120 hours, in particular 24 to 96 hours.

18. The process of any of paragraphs 1-17, wherein the fermentationproduct is recovered after fermentation, such as by distillation.

19. The process of any of paragraphs 1-18, wherein the fermentationproduct is an alcohol, preferably ethanol, especially fuel ethanol,potable ethanol and/or industrial ethanol.

19. The process of any of paragraphs 1-18, wherein the starch-containingstarting material is whole grains.

20. The process of any of paragraphs 1-19, wherein the starch-containingmaterial is derived from corn, wheat, barley, rye, milo, sago, cassava,manioc, tapioca, sorghum, rice or potatoes.

21. The process of any of paragraphs 1-20, wherein the fermentingorganism is yeast, preferably a strain of Saccharomyces, especially astrain of Saccharomyces cerevisiae.

23. The process of any of paragraphs 1-22, wherein the alpha-amylase isa bacterial alpha-amylase, in particular derived from the genusBacillus, such as a strain of Bacillus stearothermophilus, in particulara variant of a Bacillus stearothermophilus alpha-amylase, such as theone shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein.

24. The process of paragraph 23, wherein the Bacillus stearothermophilusalpha-amylase or variant thereof is truncated, preferably to be around491 amino acids long, such as from 480-495 amino acids long.

25. The process of any of paragraphs 23 or 24, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion in positionsI181+G182, and optionally a N193F substitution, or a double deletion inpositions R179+G180 (using SEQ ID NO: 1 herein for numbering).

26. The process of any of paragraphs 23-25 wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position S242,preferably a S242Q or a S242A substitution (using SEQ ID NO: 1 hereinfor numbering).

27. The process of any of paragraphs 23-26, wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position E188,preferably E188P substitution (using SEQ ID NO: 1 herein for numbering).

28. The process of any of paragraphs 1-27, wherein the alpha-amylase hasa T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 10, such as atleast 15, such as at least 20, such as at least 25, such as at least 30,such as at least 40, such as at least 50, such as at least 60, such asbetween 10-70, such as between 15-70, such as between 20-70, such asbetween 25-70, such as between 30-70, such as between 40-70, such asbetween 50-70, such as between 60-70.

29. The process of any of paragraphs 1-28, wherein the alpha-amylase isselected from the group of Bacillus stearothermophilus alpha-amylasevariants with the following mutations :I181*+G182*, and optionally N193F(using SEQ ID NO: 1 for numbering) and optionally further:

V59A + Q89R + G112D + E129V + K177L + R179E + K220P + N224L + Q254S;V59A + Q89R + E129V + K177L + R179E + H208Y + K220P + N224L + Q254S;V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E +D281N; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +I270L; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +H274K; V59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S +Y276F; V59A + E129V + R157Y + K177L + R179E + K220P + N224L + S242Q +Q254S; V59A + E129V + K177L + R179E + H208Y + K220P + N224L + S242Q +Q254S; V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S +Y276F;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + D281N;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + M284T;V59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + G416V;V59A + E129V + K177L + R179E + K220P + N224L + Q254S; V59A + E129V +K177L + R179E + K220P + N224L + Q254S + M284T; A91L + M96I + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S; E129V + K177L + R179E;E129V + K177L + R179E + K220P + N224L + S242Q + Q254S; E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + Y276F + L427M; E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + M284T; E129V + K177L + R179E +K220P + N224L + S242Q + Q254S + N376* + I377*; E129V + K177L + R179E +K220P + N224L + Q254S; E129V + K177L + R179E + K220P + N224L + Q254S +M284T; E129V + K177L + R179E + S242Q; E129V + K177L + R179V + K220P +N224L + S242Q + Q254S; K220P + N224L + S242Q + Q254S; M284V; V59A Q89R +E129V + K177L + R179E + Q254S + M284V.

30. The process of any of paragraphs 1-29, wherein the alpha-amylase isselected from the group of Bacillus stearothermophilus alpha-amylasevariants:

I181*+G182*;

I181*+G182*+N193F;

-   preferably-   I181*+G182*+E129V+K177L+R179E;-   I181*+G182*+N193F+E129V+K177L+R179E;-   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V: and

I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQID NO: 1 for numbering).

31. The process of any of paragraphs 1-30, further wherein a protease ispresent and/or added in liquefaction, wherein the protease has athermostability value of more than 25% determined as Relative Activityat 80° C./70° C.

32. The process of any of paragraphs 1-31, wherein the protease has athermostability of more than 30%, more than 40%, more than 50%, morethan 60%, more than 70%, more than 80%, more than 90%, more than 100%,such as more than 105%, such as more than 110%, such as more than 115%,such as more than 120% determined as Relative Activity at 80° C./70° C.

33. The process of any of paragraphs 1-26, wherein the protease has athermostability of between 20 and 50%, such as between 20 and 40%, suchas 20 and 30% determined as Relative Activity at 80° C./70° C.

34. The process of any of paragraphs 1-33, wherein the protease has athermostability between 50 and 115%, such as between 50 and 70%, such asbetween 50 and 60%, such as between 100 and 120%, such as between 105and 115% determined as Relative Activity at 80° C./70° C. or wherein theprotease has a thermostability of more than 10%, such as more than 12%,more than 14%, more than 16%, more than 18%, more than 20%, more than30%, more than 40%, more that 50%, more than 60%, more than 70%, morethan 80%, more than 90%, more than 100%, more than 110% determined asRelative Activity at 85° C./70° C.

35. The process of any of paragraphs 1-34, wherein the protease hasthermostability of between 10 and 50%, such as between 10 and 30%, suchas between 10 and 25% determined as Relative Activity at 85° C./70° C.

36. The process of any of paragraphs 1-35, wherein the protease has athemostability above 60%, such as above 90%, such as above 100%, such asabove 110% at 85° C. as determined using the Zein-BCA assay.

37. The process of any of paragraphs 1-36, wherein the protease has athemostability between 60-120, such as between 70-120%, such as between80-120%, such as between 90-120%, such as between 100-120%, such as110-120% at 85° C. as determined using the Zein-BCA assay.

38. The process of any of paragraphs 1-37, wherein the protease is offungal origin.

39. The process of any of paragraphs 1-38, wherein the protease is avariant of the metallo protease derived from a strain of the genusThermoascus, preferably a strain of Thermoascus aurantiacus, especiallyThermoascus aurantiacus CGMCC No. 0670, in particular the maturesequence shown in SEQ ID NO: 2.

40. The process of any of paragraphs 1-39, wherein the protease is avariant of the metallo protease disclosed as the mature part of SEQ IDNO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 inWO 2010/008841 or SEQ ID NO: 2 herein mutations selected from the groupof:

-   -   S5*+D79L+S87P+A112P+D142L;    -   D79L+S87P+A112P+T124V+D142L;    -   S5*+N26R+D79L+S87P+A112P+D142L;    -   N26R+T46R+D79L+S87P+A112P+D142L;    -   T46R+D79L+S87P+T116V+D142L;    -   D79L+P81R+S87P+A112P+D142L;    -   A27K+D79L+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+S87P+A112P+T124V+A126V+D142L;    -   D79L+S87P+A112P+D142L;    -   D79L+Y82F+S87P+A112P+D142L;    -   S38T+D79L+S87P+A112P+A126V+D142L;    -   D79L+Y82F+S87P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+A126V+D142L;    -   D79L+S87P+N98C+A112P+G135C+D142L;    -   D79L+S87P+A112P+D142L+T141C+M161C;    -   S36P+D79L+S87P+A112P+D142L;    -   A37P+D79L+S87P+A112P+D142L;    -   S49P+D79L+S87P+A112P+D142L;    -   S50P+D79L+S87P+A112P+D142L;    -   D79L+S87P+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;    -   D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;    -   S70V+D79L+Y82F+S87G+A112P+D142L;    -   D79L+Y82F+S87G+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A12P+A126V+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L,    -   A27K+D79D+Y82F+S87G+D104P+A112P+A126V+D142L,    -   A27K+Y82F+S87G+D104P+A112P+A126V+D142L.    -   A27K+D79L+Y82F+D104P+A112P+A126V+D142L;    -   A27K+Y82F+D104P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+D142L; and    -   D79L+S87P+D142L.

41. The process of any of paragraphs 1-40, wherein the protease is avariant of the metallo protease disclosed as the mature part of SEQ IDNO: 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 inWO 2010/008841 or SEQ ID NO: 2 herein with the following mutations:

D79D+S87P+A112P+D142L;

D79L+S87P+D142L; or

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

42. The process of any of paragraphs 1-41, wherein the protease varianthas at least 75% identity preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 91%,more preferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, but lessthan 100% identity to the mature part of the polypeptide of SEQ ID NO: 2disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO2010/008841 or SEQ ID NO: 2 herein.

43. The process of any of paragraphs 1-42, wherein the protease variantof the Thermoascus aurantiacus protease shown in SEQ ID NO: 2 is one ofthe following:

D79L+S87P+D142L

D79D+S87P+A112P+D142L

D79L+Y82F+S87P+A112P+D142L

S38T+D79D+S87P+A112P+A126V+D142L

D79L+Y82F+S87P+A112P+A126V+D142L

A27K+D79L+S87P+A112P+A126V+D142L

S49P+D79L+S87P+A112P+D142L

S50P+D79L+S87P+A112P+D142L

D79L+S87P+D104P+A112P+D142L

D79L+Y82F+S87G+A112P+D142L

S70V+D79L+Y82F+S87G+Y97W+A112P+D142L

D79D+Y82F+S87G+Y97W+D104P+A112P+D142L

S70V+D79L+Y82F+S87G+A112P+D142L

D79D+Y82F+S87G+D104P+A112P+D142L

D79L+Y82F+S87G+A112P+A126V+D142L

Y82F+S87G+S70V+D79L+D104P+A112P+D142L

Y82F+S87G+D79L+D104P+A112P+A126V+D142L

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

44. The process of any of paragraphs 1-43, wherein the protease is ofbacterial origin.

45. The process of any of paragraphs 1-44, wherein the protease isderived from a strain of Pyrococcus, preferably a strain of Pyrococcusfuriosus.

46. The process of any of paragraphs 1-45, wherein the protease is theone shown in SEQ ID NO: 1 in U.S. Pat. No. 6,358,726 or SEQ ID NO: 13herein.

47. The process of any of paragraphs 1-46, wherein the protease is onehaving at least 80%, such as at least 85%, such as at least 90%, such asat least 95%, such as at least 96%, such as at least 97%, such as atleast 98%, such as at least 99% identity to in SEQ ID NO: 1 in U.S. Pat.No. 6,358,726 or SEQ ID NO: 13 herein.

48. The process of any of paragraphs 1-47, further wherein acarbohydrate-source generating enzyme is present and/or added duringliquefaction step i), preferably a glucoamylase.

49. The process of any of paragraphs 1-48, wherein thecarbohydrate-source generating enzyme present and/or added duringliquefaction step i) is a glucoamylase having a heat stability at 85°C., pH 5.3, of at least 20%, such as at least 30%, preferably at least35%.

50. The process of any of paragraphs 48-49, wherein thecarbohydrate-source generating enzyme is a glucoamylase having arelative activity pH optimum at pH 5.0 of at least 90%, preferably atleast 95%, preferably at least 97%.

51. The process of any of paragraphs 48-50, wherein thecarbohydrate-source generating enzyme is a glucoamylase having a pHstability at pH 5.0 of at least at least 80%, at least 85%, at least90%.

52. The process of any of paragraphs 48-51, wherein thecarbohydrate-source generating enzyme present and/or added duringliquefaction step i) is a glucoamylase, preferably derived from a strainof the genus Penicillium, especially a strain of Penicillium oxaticumdisclosed as SEQ ID NO: 2 in WO2011/127802 or SEQ ID NO: 14 herein.

53. The process of claim 48-52, wherein the glucoamylase has at least80%, more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99% or 100% identity to the mature polypeptide shownin SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NO: 14 (mature) herein.

54. The process of any of paragraphs 48-53, wherein thecarbohydrate-source generating enzyme is a variant of the glucoamylasederived from a strain of Penicillium oxaficum disclosed as SEQ ID NO: 2in WO 2011/127802 having a K79V substitution (using the mature sequenceshown in SEQ ID NO: 14 herein for numbering).

55. The process of any of paragraphs 52-54, wherein the Penicilliumoxalicum glucoamylase has a K79V substitution (using the mature sequenceshown as SEQ ID NO: 14 herein for numbering) and further one of thefollowing:

T65A; or

Q327F; or

E501V; or

Y504T: or

Y504*; or

T65A+Q327F; or

T65A+E501V; or

T65A+Y504T; or

T65A+Y504*; or

Q327F+E501V; or

Q327F+Y504T; or

Q327F+Y504*; or

E501V+Y504T; or

E501V+Y504*; or

T65A+Q327F+E501V; or

T65A+Q327F+Y504T; or

T65A+E501V+Y504T; or

Q327F+E501V+Y504T; or

T65A+Q327F+Y504*; or

T65A+E501V+Y504*; or

Q327F+E501V+Y504*; or

T65A+Q327F+E501V+Y504T; or

T65A+Q327F+E501V+Y504*;

E501V+Y504T; or

T65A+K161S; or

T65A+Q405T; or

T65A+Q327W; or

T65A+Q327F; or

T65A+Q327Y; or

P11F+T65A+Q327F; or

R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F; or

P11F+D26C+K33C+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or

R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or

P11F+T65A+Q327W; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P11F+T65A+Q327W+E501V+Y504T; or

T65A+Q327F+E501V+Y504T; or

T65A+S105P+Q327W; or

T65A+S105P+Q327F; or

T65A+Q327W+S364P; or

T65A+Q327F+S364P; or

T65A+S103N+Q327F; or

P2N+P4S+P11F+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+D445N+V447S; or

P2N+P4S+P11F+T65A+I172V+Q327F; or

P2N+P4S+P11F+T65A+Q327F+N502*; or

P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; or

P2N+P4S+P11F+T65A+Q327F+S377T; or

P2N+P4S+P11F+T65A+V325T+Q327W; or

P2N+P4S+P11F+T65A+Q327F +D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; or

P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; or

P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; or

P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; or

P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; or

P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+Q327F+E501V+N502T +Y504*; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; or

K5A+P11F+T65A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; or

P2N+P4S+P11F+T65A+K79A+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79G+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79I+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79L+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+K79S+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or

S255N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or

P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

56. The process of any of paragraphs 48-55, further wherein aglucoamylase is present and/or added during saccharification and/orfermentation.

57. The process of any of paragraphs 1-56, wherein the glucoamylasepresent and/or added during saccharification and/or fermentation is offungal origin, preferably from a stain of Aspergillus, preferablyAspergillus niger, Aspergillus awamori, or Aspergillus oryzae; or astrain of Trichoderma, preferably T. reesei; or a strain of Talaromyces,preferably T. emersonii, or a strain of Pycnoporus, or a strain ofGloephyllum, or a strain of the Nigrofomes.

58. The process of any of paragraphs 1-57, further wherein a pullulanaseis present and/or added during liquefaction and/or saccharification.

59. The process of any of paragraphs 1-58, further wherein anendoglucnase is present and/or added during liquefaction step i).

60. The process of paragraphs 59, wherein the endoglucnase is the oneshown as SEQ ID NO: 2 in WO 2013/019780 or in SEQ ID NO: 9 herein, e.g.,derived from a strain of Talaromyces leycettanus, or an endoglucanasehaving at least 60%, such as at least 70%, such as at least 75%identity, preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 91%, more preferablyat least 92%, even more preferably at least 93%, most preferably atleast 94%, and even most preferably at least 95%, such as even at least96%, at least 97%, at least 98%, at least 99%, such as 100% identity tothe mature part of the polypeptide of SEQ ID NO: 9 herein.

61. The process of paragraphs 1-59, wherein a phytase is present and/oradded during liquefaction and/or saccharification.

62. The process of any of paragraphs 1-61, comprising the steps of:

-   -   i) liquefying the starch-containing material at a temperature in        the range from 70-100° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus;        -   a hemicellulase, in particular a xylanase, having a Melting            Point (DSC) above 80° C., preferably above 85° C.,            especially above 90° C., in particular above 95° C.;    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism.

63. A process of paragraphs 1-62, comprising the steps of:

-   -   i) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase, preferably derived from Bacillus            stearothermophilus, having a T½ (min) at pH 4.5, 85° C.,            0.12 mM CaCl₂ of at least 10;        -   a hemicellulase, in particular a xylanase, having a Melting            Point (DSC) above 80° C.; preferably above 85° C.,            especially above 90° C., in particular above 95° C.;        -   optionally an endoglucanase, in particular one having a            Melting Point (DSC) above 70° C., preferably above 75° C.;    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism.

64. A process of paragraphs 1-63, comprising the steps of:

-   -   i) liquefying the starch-containing material at a temperature        between 70-100° C. using:        -   a bacterial alpha-amylase, preferably derived from Bacillus            stearothermophilus, having a T½ (min) at pH 4.5, 85° C.,            0.12 mM CaCl₂ of at least 10;        -   a hemicellulase, in particular a xylanase, having a Melting            Point (DSC), above 80° C., preferably above 85° C.,            especially above 90° C., in particular above 95° C.;        -   optionally a protease, preferably derived from Pyrococcus            furiosus or Thermoascus aurantiacus, having a            thermostability value of more than 20% determined as            Relative Activity at 80° C./70° C.;        -   optionally an endoglucanase, in particular one having a            Melting Point (DSC) above 70° C.;    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism.

65. A process of paragraphs 1-64, comprising the steps of:

-   -   i) liquefying the starch-containing material at a pH in the        range between from above 4.0-6.5 at a temperature above the        initial gelatinization temperature using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182, and optional            substitution N193F; and optionally further comprising one of            the following set of substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1            herein for numbering);        -   a hemicellulase, in particular a xylanase, having a Melting            Point (DSC) above 80° C., preferably above 85° C.,            especially above 90° C., in particular above 95° C.; in            particular a xylanase having at least 60%, such as at least            70%, such as at least 75% identity, preferably at least 80%,            more preferably at least 85%, more preferably at least 90%,            more preferably at least 91%, more preferably at least 92%,            even more preferably at least 93%, most preferably at least            94%, and even most preferably at least 95%, such as even at            least 96%, at least 97%, at least 98%, at least 99% to the            mature part of any of the polypeptides shown in SEQ ID NOs:            3, 4, 5, 8 and 34 herein;        -   optionally a protease having a thermostability value of more            than 20% determined as Relative Activity at 80° C./70° C.,            in particular derived from Pyrococcus furiosus and/or            Thermoascus aurantiacus; and optionally        -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO:            14 herein having substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; (using SEQ ID NO: 14 hereinfor            numbering);        -   optionally an endoglucanase, in particular one having a            Melting Point (DSC) above 70° C., preferably above 75° C.,            especially above 80° C.;        -   ii) saccharifying using a glucoamylase enzyme;        -   iii) fermenting using a fermenting organism.

66. A process of paragraphs 1-65, comprising the steps of:

-   -   i) liquefying the starch-containing material at a temperature        between 70-100° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182, and optional            substitution N193F; and optionally further comprising one of            the following set of substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1            herein for numbering);        -   a hemicellulase, preferably xylanase, having a Melting Point            (DSC), above 80° C., preferably above 85° C., especially            above 90° C., in particular above 95° C.; in particular a            xylanase having at least 80% identity to the mature part of            the polypeptide of SEQ ID NOs: 3, 4, 5, 8 and 34 herein;        -   optionally an endoglucanase, in particular an having a            Melting Point (DSC) above 70° C., preferably above 75° C.,            especially above 80° C.;        -   optionally a protease having a thermostability value of more            than 20% determined as Relative Activity at 80° C./70° C.,            in particular a protease derived from Pyrococcus furiosus            and/or Thermoascus aurantiacus; and        -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO:            14 herein comprising substitutions selected from the group            of:        -   K79V; or        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for            numbering);        -   ii) saccharifying using a glucoamylase enzyme;        -   iii) fermenting using a fermenting organism.

67. A process of paragraphs 1-65, comprising the steps of:

-   -   i) liquefying the starch-containing material at a temperature        between 70-100° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182, and optional            substitution N193F; and optionally further comprising one of            the following set of substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   V59A Q89R+E129V+K177L+R179E+Q254S+M284V;        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO:            1 herein for numbering);        -   a hemicellulase, in particular a xylanase, having a Melting            Point (DSC) above 80° C., preferably above 85° C.,            especially above 90° C., in particular above 95° C.; in            particular a xylanase having at least 60%, such as at least            70%, such as at least 75% identity, preferably at least 80%,            more preferably at least 85%, more preferably at least 90%,            more preferably at least 91%, more preferably at least 92%,            even more preferably at least 93%, most preferably at least            94%, and even most preferably at least 95%, such as even at            least 96%, at least 97%, at least 98%, at least 99% to the            mature part of any of the polypeptides shown in SEQ ID NOs:            3, 4, 5, 8 and 34 herein;        -   optionally an endoglucanase having a Melting Point (DSC),            above 70° C., preferably above 75° C., especially above 80°            C.;        -   optionally a protease having a thermostability value of more            than 20% determined as Relative Activity at 80° C./70° C.,            in particular one derived from Pyrococcus furiosus;        -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO:            14 herein having substitutions selected from the group of:        -   K79V; or        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P45+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);        -   optionally an endoglucanase in particular one having a            Melting Point (DSC) above 70° C., preferably above 75° C.,            especially above 80° C.;        -   ii) saccharifying using a glucoamylase enzyme;        -   iii) fermenting using a fermenting organism.

68. The process of any of paragraphs 62-67, wherein the Bacillusstearothermophilus alpha-amylase (SEQ ID NO: 1 herein) is the maturealpha-amylase or corresponding mature alpha-amylases having at least60%, such as at least 70%, such as at least 80% identity, such as atleast 90% identity, at least 95% identity at least 96% identity at least97% identity at least 99% identity to the SEQ ID NO: 1 herein.

69. The process of any of paragraphs 64-68, wherein the Pyrococcusfuriosus protease (SEQ ID NO: 13 herein) or Thermoascus aurantiacusprotease (SEQ ID NO: 3) is the mature protease or corresponding matureprotease having at least 80% identity, at least 90% identity, at least95% identity at least 96% identity at least 97% identity at least 99%identity to SEQ ID NO: 13 herein or SEQ ID NO: 3 herein, respectively.

70. The process of any of paragraphs 65-69, wherein the Penicilliumoxalicum glucoamylase (SEQ ID NO: 14 herein), or a variant thereof, isthe mature glucoamylase or corresponding mature glucoamylase having atleast 80% identity, at least 90% identity, at least 95% identity atleast 96% identity at least 97% identity at least 99% identity to theSEQ ID NO: 14 herein.

71. The process of any of paragraphs 1-70, wherein cellulase orcellulolytic enzyme composition is present or adding during fermentationor simultaneous saccharification and fermentation.

72. The process of any of paragraphs 1-71, wherein a cellulase orcellulolytic enzyme composition and a glucoamylase are present or addedduring fermentation or simultaneous saccharification and fermentation.

73. The process of any of paragraphs 1-72, wherein cellulase orcellulolytic enzyme composition and glucoamylase present or added duringfermentation or simultaneous saccharification and fermentation added.

74. The process of any of paragraphs 71-73 wherein the cellulase orcellulolytic enzyme composition is derived from Trichoderma reesei,Humicola insolens, Chrysosporium lucknowense or Penicillium decumbens.

75. The process of any of paragraphs 71-74, wherein the cellulase orcellulolytic enzyme composition comprises a beta-glucosidase, acellobiohydrolase, and an endoglucanase.

76. The process of any of paragraphs 71-75, wherein the cellulase orcellulolytic enzyme composition comprises a beta-glucosidase, preferablyone derived from a strain of the genus Aspergillus, such as Aspergillusoryzae, such as the one disclosed in WO 2002/095014 or the fusionprotein having beta-glucosidase activity disclosed in WO 2008/057637, orAspergillus fumigatus, such as one disclosed in SEQ ID NO: 2 in WO2005/047499 or SEQ ID NO: 29 herein or an Aspergillus fumigatusbeta-glucosidase variant disclosed in WO 2012/044915; or a strain of thegenus a strain Penicillium, such as a strain of the Penicilliumbrasilianum disclosed in WO 2007/019442, or a strain of the genusTrichoderma, such as a strain of Trichoderma reesei.

77. The process of any of paragraphs 71-76, wherein the cellulase orcellulolytic enzyme composition comprises a GH61 polypeptide havingcellulolytic enhancing activity such as one derived from the genusThermoascus, such as a strain of Thermoascus aurantiacus, such as theone described in WO 2005/074656 or SEQ ID NO: 30 herein; or one derivedfrom the genus Thielavia, such as a strain of Thielavia terrestris, suchas the one described in WO 2005/074647 as SEQ ID NO: 7; or one derivedfrom a strain of Aspergillus, such as a strain of Aspergillus fumigatus,such as the one described in WO 2010/138754 as SEQ ID NO: 1; or onederived from a strain derived from Penicillium, such as a strain ofPenicillium emersonii, such as the one disclosed in WO 2011/041397 asSEQ ID NO: 2 or SEQ ID NO: 31 herein.

78. The process of any of paragraphs 71-77, wherein the cellulase orcellulolytic enzyme composition comprises a cellobiohydrolase I (CBH I),such as one derived from a strain of the genus Aspergillus, such as astrain of Aspergillus fumigatus, such as the Cel7a CBH I disclosed inSEQ ID NO: 6 in WO 2011/057140 or SEQ ID NO: 32 herein, or a strain ofthe genus Trichoderma, such as a strain of Trichoderma reesei.

79. The process of any of paragraphs 71-78, wherein the cellulase orcellulolytic enzyme composition comprises a cellobiohydrolase II (CBHII), such as one derived from a strain of the genus Aspergillus, such asa strain of Aspergillus fumigatus; such as the one disclosed as SEQ IDNO: 33 herein or a strain of the genus Trichoderma, such as Trichodermareesei, or a strain of the genus Thielavia, such as a strain ofThielavia terrestris, such as cellobiohydrolase II CEL6A from Thielaviaterrestris.

80. The process of any of paragraphs 71-79, wherein the cellulase orcellulolytic enzyme composition comprises a GH61 polypeptide havingcellulolytic enhancing activity and a beta-glucosidase.

81. The process of any of paragraphs 71-80, wherein the cellulase orcellulolytic enzyme composition comprises a GH61 polypeptide havingcellulolytic enhancing activity and a beta-glucosidase.

82. The process of any of paragraphs 71-81, wherein the cellulase orcellulolytic enzyme composition comprises a GH61 polypeptide havingcellulolytic enhancing activity, a beta-glucosidase, and a CBH I.

83. The process of any of paragraphs 71-82, wherein the cellulase orcellulolytic enzyme composition comprises a GH61 polypeptide havingcellulolytic enhancing activity, a beta-glucosidase, a CBH I, and a CBHII.

84. The process of any of paragraphs 71-83, wherein the cellulase orcellulolytic enzyme composition is a Trichoderma reesei cellulolyticenzyme composition, further comprising Thermoascus aurantiacus GH61Apolypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO2005/074656 or SEQ ID NO: 30 herein), and Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

85. The process of any of paragraphs 71-84, wherein the cellulase orcellulolytic enzyme composition is a Trichoderma reesei cellulolyticenzyme composition, further comprising Thermoascus aurantiacus GH61Apolypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO2005/074656 or SEQ ID NO: 30 herein) and Aspergillus fumigatusbeta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 29herein).

86. The process of any of paragraphs 71-85, wherein the cellulase orcellulolytic enzyme composition is a Trichoderma reesei cellulolyticenzyme composition further comprising Penicillium emersonii GH61Apolypeptide having cellulolytic enhancing activity disclosed in WO2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 31 herein; and Aspergillusfumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) or SEQ IDNO: 29 herein or a variant thereof with one or more, such as all, of thefollowing substitutions: F100D, S283G, N456E, F512Y.

87. The process of any of paragraphs 71-86, wherein the cellulase orcellulolytic enzyme composition comprises one or more of the followingcomponents

(i) an Aspergillus fumigatus cellobiohydrolase I;

(ii) an Aspergillus fumigatus cellobiohydrolase II;

(iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and

(iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancingactivity; or homologs thereof.

88. The process of any of paragraphs 71-87, wherein the cellulase orcellulolytic enzyme composition is SPIRIZYME ACHIEVE™, CELLIC CTEC™,CELLIC CTEC2™, CELLIC CTEC3™, ACCELLERASE 1000™, ACCELLERASE 1500™,ACCELLERASE DUET™, ACCELLERASE TRIO™.

89. The process of any of paragraphs 71-88, wherein the glucoamylasepresent or added during saccharification or simultaneoussaccharification and fermentation is of fungal origin, preferably from astain of Aspergillus, preferably Aspergillus niger, Aspergillus awamori,or Aspergillus oryzae: or a strain of Trichoderma, preferably T. reesei;or a strain of Talaromyces, preferably T. emersonii, or a strain ofPycnoporus, or a strain of Gloephyllum, or a strain of the Nigrofomes.

90. The process of any of paragraphs 72-89, wherein the glucoamylase isa blend of glucoamylase derived from Talaromyces emersonii disclosed inWO 99/28448 or SEQ ID NO: 28 herein, Trametes cingulata glucoamylasedisclosed as SEQ ID NO: 2 in WO 06/69289 or SEQ ID NO: 26 herein, andRhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylaselinker and SBD disclosed as V039 in Table 5 in WO 2006/069290 or SEQ IDNO: 27 herein.

91. The process of any of paragraphs 72-90, wherein the alpha-amylase isthe Rhizomucor pusillus alpha-amylase having an Aspergillus nigerglucoamylase linker and starch-binding domain (SBD) (SEQ ID NO: 27herein) which further comprises at least one of the followingsubstitutions or combinations of substitutions: D165M; Y141W; Y141R;K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W;G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R;Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N;Y141W+K192R V410A: G128D+Y141W+D143N; Y141W+D143N+P2190;Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P2190;G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ IDNO: 27 herein for numbering).

92. A composition comprising:

-   -   an alpha-amylase;    -   a hemicellulase, in particular a xylanase, having a Melting        Point (DSC) above 80° C., preferably above 85° C., especially        above 90° C., in particular above 95° C.;    -   optionally an endoglucanase;    -   optionally a protease;    -   optionally a carbohydrate-source generating enzyme.

93. The composition of paragraph 92, wherein the alpha-amylase is abacterial or fungal alpha-amylase.

94. The composition of any of paragraphs 92-93, wherein thealpha-amylase is a bacterial alpha-amaylase, in particular from thegenus Bacillus, such as a strain of Bacillus stearothermophilus, inparticular a variant of a Bacillus stearothermophilus alpha-amylase,such as the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1herein.

95. The composition of paragraph 94, wherein the Bacillusstearothermophilus alpha-amylase or variant thereof is truncated,preferably to be around 491 amino acids long, such as from 480-495 aminoacids long.

96. The composition of any of paragraphs 92-95, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion in positionsI181+G182, and optionally a N193F substitution, or a double deletion inpositions R179+G180 (using SEQ ID NO: 1 herein for numbering).

97. The composition of any of paragraphs 92-96 wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position S242,preferably S2420 substitution (using SEQ ID NO: 1 herein for numbering),

98. The composition of any of paragraphs 92-97, wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position E188,preferably E188P substitution (using SEQ ID NO: 1 herein for numbering).

99. The composition of any of paragraphs 92-98, wherein thealpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of atleast 10, such as at least 15, such as at least 20, such as at least 25,such as at least 30, such as at least 40, such as at least 50, such asat least 60, such as between 10-70, such as between 15-70, such asbetween 20-70, such as between 25-70, such as between 30-70, such asbetween 40-70, such as between 50-70, such as between 60-70.

100. The composition of any of paragraphs 92-99, wherein thealpha-amylase is selected from the group of Bacillus stearomthermphilusalpha-amylase variants shown in SEQ ID NO: 1 herein having a doubledeletion in I181*+G182*, and optionally a subsitution in N193F, inparticular further having one of the following set of substitutions:

E129V+K177L+R179E;

V59A+Q89R+E129V+K177L+R179E;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and

E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1 herein fornumbering).

101, The composition of any of paragraphs 92-100, wherein thehemicellulase, in particular xylanase, such as GH10 xylanase or GH11xylanase, has a Melting Point (DSC) above 82° C., such as above 84° C.,such as above 86° C., such as above 88° C., such as above 90° C., suchas above 92° C., such as above 94° C., such as above 96° C., such asabove 98° C., such as above 100° C.

102. The composition of any of paragraphs 92-101, wherein thehemicellulase, in particular xylanase, especially GH10 or GH11 xylanasehas at least 60%, such as at least 70%, such as at least 75%, preferablyat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, evenmore preferably at least 93%, most preferably at least 94%, and evenmost preferably at least 95%, such as even at least 96%, at least 97%,at least 98%, at least 99%, such as 100% identity to the mature part ofthe polypeptide of SEQ ID NO: 3 herein (GH10 xylanase) or SEQ ID NO: 34(GH11 xylanase), preferably derived from a strain of the genusDictyoglomus, such as a strain of Dictyogllomus thermophilum.

103. The composition of any of paragraphs 92-101, wherein thehemicellulase, in particular xylanase, especially GH10 xylanase has atleast 60%, such as at least 70%, such as at least 75% identity,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, such as 100% identity to themature part of the polypeptide of SEQ ID NO: 4 herein, preferablyderived from a strain of the genus Rasamsonia, such as a strain ofRasomsonia byssochlamydoides.

104. The composition of any of paragraphs 92-101, wherein thehemicellulase, in particular xylanase, especially GH10 xylanase has atleast 60%, such as at least 70%, such as at least 75% identity,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, such as 100% identity to themature part of the polypeptide of SEQ ID NO: 5 herein, preferablyderived from a strain of the genus Talaromyces, such as a strain ofTalaromyces leycettanus.

105. The composition of any of paragraphs 92-101, wherein thehemicellulase, in particular xylanase, especially GH10 xylanase has atleast 60%, such as at least 70%, such as at least 75% identity,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, such as 100% identity to themature part of the polypeptide of SEQ ID NO: 8 herein, preferablyderived from a strain of the genus Aspergillus, such as a strain ofAspergillus fumigatus.

106. The composition of any of paragraphs 92-105, comprising

-   -   an alpha-amylase;    -   a hemicellulase having a Melting Point (DSC) above 80° C.        preferably above 85° C., especially above 90° C., in particular        above 95° C.;    -   an endoglucanase having Melting Point (DSC) above 70° C.,        preferably above 75° C., especially above 80° C., in particular        above 85° C.

107. The composition of any of paragraphs 92-106, wherein the proteasewith a thermostability value of more than 20%, such as more than 25%determined as Relative Activity at 80° C./70° C.

108. The composition of any of paragraphs 92-107, wherein the proteasehas a thermostability of more than 30%, more than 40%, more than 50%,more than 60%, more than 70%, more than 80%, more than 90%, more than100%, such as more than 105%, such as more than 110%, such as more than115%, such as more than 120% determined as Relative Activity at 80°C./70° C.

109. The composition of any of paragraphs 92-108, wherein the proteasehas a thermostability of between 20 and 50%, such as between 20 and 40%,such as 20 and 30% determined as Relative Activity at 8° C./70° C.

110. The composition of any of paragraphs 92-109, wherein the proteasehas a thermostability between 50 and 115%, such as between 50 and 70%,such as between 50 and 60%, such as between 100 and 120%, such asbetween 105 and 115% determined as Relative Activity at 80° C./70° C.

111. The composition of any of paragraphs 92-110, wherein the proteasehas a thermostability of more than 10%, such as more than 12%, more than14%, more than 16%, more than 18%, more than 20%, more than 30%, morethan 40%, more that 50%, more than 60%, more than 70%, more than 80%,more than 90%, more than 100%, more than 110% determined as RelativeActivity at 85° C./70° C.

112. The composition of any of paragraphs 92-111, wherein the proteasehas thermostability of between 10 and 50%, such as between 10 and 30%,such as between 10 and 25% determined as Relative Activity at 85° C./70°C.

113. The composition of any of paragraphs 92-112, wherein the proteasehas a themostability above 60%, such as above 90%, such as above 100%,such as above 110% at 85° C. as determined using the Zein-BCA assay.

114. The composition of any of paragraphs 92-113, wherein the proteasehas a themostability between 60-120, such as between 70-120%, such asbetween 80-120%, such as between 90-120%, such as between 100-120%, suchas 110-120% at 85° C. as determined using the Zein-BCA assay.

115. The composition of any of paragraphs 92-114, wherein the proteaseis of fungal origin.

116. The composition of any of paragraphs 92-115, wherein the proteaseis a variant of the metallo protease derived from a strain of the genusThermoascus, preferably a strain of Thermoascus aurantiacus, especiallyThermoascus aurantiacus CGMCC No. 0670, in particular the one shown inSEQ ID NO: 2 herein.

117. The composition of any of paragraphs 92-116, wherein the proteaseis a variant of the protease disclosed as the mature part of SEQ ID NO:2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO2010/008841 or SEQ ID NO: 2 herein with the following mutations:

D79L+S87P+A112P+D142L:

D79L+S87P+D142L; or

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L (using SEQ ID NO: 2 hereinfor numbering).

118. The composition of any of paragraphs 92-118, wherein the proteasevariant has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or the maturepart of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 2 herein.

119. The composition of any of paragraphs 92-118, wherein the proteasevariant of the Thermoascus aurantiacus protease shown in SEQ ID NO: 2herein is one of the following:

D79L+S87P+D142L

D79L+S87P+A112P+D142L

D79L+Y82F+S87P+A112P+D142L

S38T+D79L+S87P+A112P+A126V+D142L

D79L+Y82F+S87P+A112P+A126V+D142L

A27K+D79D+S87P+A112P+A126V+D142L

S49P+D79L+S87P+A112P+D142L

S50P+D79L+S87P+A112P+D142L

D79L+S87P+D104P+A112P+D142L

D79L+Y82F+S87G+A112P+D142L

S70V+D79D+Y82F+387G+Y97W+A112P+D142L

D79L+Y82F+S87G+Y97W+D104P+A112P+D142L

S70V+D79L+Y82F+S87G+A112P+D142L

D79L+Y82F+S87G+D104P+A112P+D142L

D79L+Y82F+S87G+A112P+A126V+D142L

Y82F+S87G+S70V+D79L+D104P+A112P+D142L

Y82F+S87G+D79L+D104P+A112P+A126V+D142L

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

120. The composition of any of paragraphs 92-119, wherein the proteaseis of bacterial origin.

121. The composition of any of paragraphs 92-120, wherein the proteaseis derived from a strain of Pyrococcus, preferably a strain ofPyrococcus furiosus.

122. The composition of any of paragraphs 92-121, wherein the proteaseis the one shown in SEQ ID NO: 1 in U.S. Pat. No. 6,358,726 or SEQ IDNO: 13 herein.

123. The composition of any of paragraphs 92-122, wherein the proteaseis one having at least 80%, such as at least 85%, such as at least 90%,such as at least 95%, such as at least 96%, such as at least 97%, suchas at least 98%, such as at least 99% identity to SEQ ID NO: 1 in U.S.Pat. No. 6,358,726 or SEQ ID NO: 13 herein.

124. The composition of any of paragraphs 92-123, wherein acarbohydrate-source generating enzyme is a glucoamylase.

125. The composition of any of paragraphs 92-124, wherein thecarbohydrate-source generating enzyme is a glucoamylase having a heatstability at 85° C., pH 5.3, of at least 20%, such as at least 30%,preferably at least 35%.

126. The composition of any of paragraphs 92-125, wherein thecarbohydrate-source generating enzyme is a glucoamylase having arelative activity pH optimum at pH 5.0 of at least 90%, preferably atleast 95%, preferably at least 97%.

127. The composition of any of paragraphs 92-126, wherein thecarbohydrate-source generating enzyme is a glucoamylase having a pHstability at pH 5.0 of at least at least 80%, at least 85%, at least90%.

128. The composition of any of paragraphs 92-127, wherein thecarbohydrate-source generating enzyme is a glucoamylase, preferablyderived from a strain of the genus Penicillium, especially a strain ofPenicillium oxalicum disclosed as SEQ ID NO: 2 in WO 2011/127802 or SEQID NO: 14 herein.

129. The composition of paragraphs 92-128, wherein the glucoamylase hasat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, evenmore preferably at least 93%, most preferably at least 94%, and evenmost preferably at least 95%, such as even at least 96%, at least 97%,at least 98%, at least 99% or 100% identity to the mature polypeptideshown in SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NO: 14 herein.

130. The composition of any of paragraphs 92-129, wherein thecarbohydrate-source generating enzyme is a variant of the glucoamylasederived from a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2in WO 2011/127802 having a K79V substitution (using the mature sequenceshown in SEQ ID NO: 14 for numbering),

131. The composition of any of paragraphs 92-130, further comprising apullulanase, in particular a pullulanase of family GH57, wherein thepullulanase preferably includes an X47 domain as disclosed in WO2011/087836.

132. The composition of any of paragraphs 92-131, further comprising aphytase.

133. The composition of paragraph 132, wherein the phytase is derivedfrom Buttiauxelia, such as Buttiauxella gaviniae, Buttiauxella agrestis,or Buttiauxella noackies disclosed in WO 2008/092901, or Citrobacterbraakii, such as one disclosed in WO 2006/037328.

134. The composition of any of paragraphs 92-136 comprising

-   -   an alpha-amylase derived from Bacillus stearothermophiius;    -   a hemicellulase, in particular a xylanase, having a Melting        Point (DSC) above 80° C., preferably above 85° C., especially        above 90° C., in particular above 95° C.;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.        derived from Pyrococcus furiosus or Thermoascus aurantiacus; and    -   optionally an endoglucanase in particular one having a Melting        Point (DSC) above 70° C., preferably above 75° C., especially        above 80° C., in particular above 85° C.; in particular a        xylanase having at least 60%, such as at least 70%, such as at        least 75% identity, preferably at least 80%, more preferably at        least 85%, more preferably at least 90%, more preferably at        least 91%, more preferably at least 92%, even more preferably at        least 93%, most preferably at least 94%, and even most        preferably at least 95%, such as even at least 96%, at least        97%, at least 98%, at least 99% to the mature part of any of the        polypeptides shown in SEQ ID NOs: 9, 35, 36, 37 and 38 herein;    -   optionally a glucoamylase, such as one derived from Penicillium        oxalicum.

135. The composition of any of paragraphs 92-134, comprising

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   a hemicellulase, in particular a xylanase, having a Melting        Point (DSC) above 80° C. preferably above 75° C., especially        above 80° C., in particular above 85° C.;    -   optionally an endoglucanase, in particular one having a Melting        Point (DSC) above 70° C., preferably above 75° C., especially        above 80° C.; in particular above 85° C.; in particular a        xylanase having at least 60%, such as at least 70%, such as at        least 75% identity, preferably at least 80%, more preferably at        least 85%, more preferably at least 90%, more preferably at        least 91%, more preferably at least 92%, even more preferably at        least 93%, most preferably at least 94%, and even most        preferably at least 95%, such as even at least 96%, at least        97%, at least 98%, at least 99% to the mature part of any of the        polypeptides shown in SEQ ID NOs: 9, 35, 36, 37 or 38 herein;    -   optionally a protease, preferably derived from Pyrococcus        furiosus or Thermoascus aurantiacus, having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.; and    -   optionally a glucoamylase, e.g., derived from Penicillium        oxalicum.

136. The composition of any of paragraphs 92-135, comprising

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and optionally substitution N193F;        and optionally further one of the following set of        substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering);    -   a hemicellulase, in particular a xylanase having a Melting Point        (DSC) above 70° C., preferably above 75° C., in particular above        80° C., especially above 85° C.; in particular a xylanase having        at least 60%, such as at least 70%, such as at least 75%        identity, preferably at least 80%, more preferably at least 85%,        more preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99% to the mature part of any of the polypeptides shown in SEQ        ID NOs: 3, 4, 5, 8 and 34 herein;    -   optionally an endoglucanase, in particular one having a Melting        Point (DSC) above 70° C., preferably above 75° C., especially        above 80° C., in particular above 85° C.; in particular a        xylanase having at least 60%, such as at least 70%, such as at        least 75% identity, preferably at least 80%, more preferably at        least 85%, more preferably at least 90%, more preferably at        least 91%, more preferably at least 92%, even more preferably at        least 93%, most preferably at least 94%, and even most        preferably at least 95%, such as even at least 96%, at least        97%, at least 98%, at least 99% to the mature part of any of the        polypeptides shown in SEQ ID NOs: 9, 35, 36, 37 and 38 herein;    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus, having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C.; and    -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 14        herein having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 herein for        numbering),

137. The composition of any of paragraphs 92-136, wherein thealpha-amylase is a bacterial alpha-amylase in particular derived fromBacillus stearothermophllus (SEQ ID NO: 1 herein), or a variant thereof,is the mature alpha-amylase or corresponding mature alpha-amylaseshaving at least 80% identity, at least 90% identity, at least 95%identity at least 96% identity at least 97% identity at least 99%identity to the SEQ ID NO: 1 herein.

138. Use of a composition of paragraphs 92-137 for liquefying astarch-containing material. hemicellulase, in particular xylanase, inliquefaction in a fermentation product production process.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.Various references are cited herein, the disclosures of which areincorporated by reference in their entireties. The present invention isfurther described by the following examples which should not beconstrued as limiting the scope of the invention.

Materials & Methods

Materials:

Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylasewith the mutations:I181*1+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to491 amino acids (SEQ ID NO: 1 herein). Endoglucanase TL (EG TL):Endoglucoanase GH5 from Talaromyces leycettanus disclosed inWO2013/019780 as SEQ ID NO: 2 and SEQ ID NO: 9 herein. (P23YSQ).

Xylalanase TL (TI Xyl): Family GH10 xylanase from Talaromycesleycettanus shown in SEQ ID NO: 5 herein.

Xylanase DT (Dt Xyl): Family GH11 xylanase from Dictyoglomusthermophilum shown in SEQ ID NO: 34 herein.

Endoglucanase PC (EG PC): Endoglucanase GH5 from Penicillium capsulatumdisclosed as SEQ ID NO: 35 herein. (P244HZ)

Endoglucanase TS (EG TS): Endoglucanase GH5 from Trichophaea saccatadisclosed as SEQ ID NO: 36 herein. (P2PJ)

Endoglucanase TT (EG TT): Endoglucoanase GH45 from Thielavia terrestrisdisclosed in SEQ ID NO: 37 herein. (P24PYU)

Endoglucanase SF (EG SF): Endoglucanase GH45 from Sordaria fimicoladisclosed in co-pending application PCT/CN2012/080220 as SEQ ID NO: 2and SEQ IDNO: 38 herein (P2CF).

Protease Pfu: Protease derived from Pyrococcus furiosus purchased fromTakara Bio (Japan) as Pfu Protease S (activity 10.5 mg/mL) and alsoshown in SEQ ID NO: 13 herein.

Glucoamylase Po: Mature part of the Penicillium oxalicum glucoamylasedisclosed as SEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 14herein.

Glucoamylase PE001: Variant of the Penicillium oxalicum glucoamylasehaving a K79V substitution using the mature sequence shown in SEQ ID NO:14 for numbering.

Protease Pfu: Protease derived from Pyrococcus furiosus shown in SEQ IDNO: 13 herein.

Glucoamylase Po 498 (GA498): Variant of Penicillium oxalicumglucoamylase having the following mutations:K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 14 for numbering).

Glucoamylase SA (GSA): Blend comprising Talaromyces emersoniiglucoamylase disclosed as SEQ ID NO: 34 in WO99/28448 or SEQ ID NO: 28herein, Trametes cingulata glucoamylase disclosed as SEQ ID NO: 2 in WO06/69289 or SEQ ID NO: 26 herein, and Rhizomucor pusillus alpha-amylasewith Aspergillus niger glucoamylase linker and starch binding domain(SBD) disclosed in SEQ ID NO: 27 herein having the followingsubstitutions G128D+D143N (activity ratio in AGU:AGU:FAU-F is about20:5:1).

Protease X: Metallo protease derived from Thermoascus aurantiacus CGMCCNo. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 2 herein and aminoacids 1-177 in SEQ ID NO: 2 in WO 2003/048353

Yeast: ETHANOL RED™ available from Red Star/Lesaffre, USA.

Methods

Determination of Td by Differential Scanning calorimetry forEndoglucanases and Hemicellulases.

The thermostability of an enzyme is determined by Differential Scanningcalorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter(MicroCal Inc., Piscataway, N.J., USA). The thermal denaturationtemperature, Td (° C.), is taken as the top of denaturation peak (majorendothermic peak) in thermograms (Cp vs. T) obtained after heatingenzyme solutions (approx. 0.5 mg/ml) in buffer (50 mM acetate, pH 5.0)at a constant programmed heating rate of 200 K/hr.

Sample- and reference-solutions (approx. 0.2 ml) are loaded into thecalorimeter (reference: buffer without enzyme) from storage conditionsat 10° C. and thermally pre-equilibrated for 20 minutes at 20° C. priorto DSC scan from 20° C. to 120° C. Denaturation temperatures aredetermined at an accuracy of approximately +/−1° C.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention the degree of identity between twoamino acid sequences, as well as the degree of identity between twonucleotide sequences, may be determined by the program “align” which isa Needleman-Wunsch alignment (i.e. a global alignment). The program isused for alignment of polypeptide, as well as nucleotide sequences. Thedefault scoring matrix BLOSUM50 is used for polypeptide alignments, andthe default identity matrix is used for nucleotide alignments. Thepenalty for the first residue of a gap is −12 for polypeptides and −16for nucleotides. The penalties for further residues of a gap are −2 forpolypeptides, and −4 for nucleotides.

“Align” is part of the FASTA package version v20u6 (see W. R. Pearsonand D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA,” Methods inEnzymology 183:63-98). FASTA protein alignments use the Smith-Watermanalgorithm with no limitation on gap size (see “Smith-Watermanalgorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biol.147:195-197).

Protease Assays AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended inBorax/NaH₂PO₄ buffer pH9 while stirring. The solution is distributedwhile stirring to microtiter plate (100 microL to each well), 30 microLenzyme sample is added and the plates are incubated in an EppendorfThermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzymesample (100° C. boiling for 20 min) is used as a blank. After incubationthe reaction is stopped by transferring the microtiter plate onto iceand the coloured solution is separated from the solid by centrifugationat 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant istransferred to a microtiter plate and the absorbance at 595 nm ismeasured using a BioRad Microplate Reader.

pNA-Assay

50 microL protease-containing sample is added to a microtiter plate andthe assay is started by adding 100 microL 1 mM pNA substrate (5 mgdissolved in 100 microL DMSO and further diluted to 10 mL withBorax/NaH₂PO₄ buffer pH 9.0). The increase in OD₄₀₅ at room temperatureis monitored as a measure of the protease activity.

Glucoamylase Activity (AGU)

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU-A)

Alpha amylase activity is measured in KNU(A) Kilo Novozymes Units (A),relative to an enzyme standard of a declared strength.

Alpha amylase in samples and α-glucosidase in the reagent kit hydrolyzethe substrate (4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside(ethylidene-G₇PNP) to glucose and the yellow-colored p-nitrophenol.

The rate of formation of p-nitrophenol can be observed by Konelab 30.This is an expression of the reaction rate and thereby the enzymeactivity.

The enzyme is an alpha-amylase pith the enzyme classification number EC3.2.1.1.

Parameter Reaction conditions Temperature 37° C. pH 7.00 (at 37° C.)Substrate conc. Ethylidene-G₇PNP, R2: 1.86 mM Enzyme conc. 1.35-4.07KNU(A)/L (conc. of high/low standard in reaction mixture) Reaction time2 min Interval kinetic measuring 7/18 sec. time Wave length 405 nm Conc.of reagents/chemicals α-glucosidase, R1: ≥3.39 kU/L critical for theanalysis

A folder EB-SM-5091.02-D on determining KNU-A actitvity is availableupon request to Novozymes A/S, Denmark, which folder is hereby includedby reference.

Alpha-Amylase Activity KNU(S)

BS-amylase in samples and the enzyme alpha-glucosidase in the reagentkit hydrolyze substrate(4,6-ethylidene(G7)-p-nitrophenyl(G1)-alpha-D-maltoheptaoside(ethylidene-G7PNP)) to glucose and the yellow-colored p-nitrophenol.

The rate of formation of p-nitrophenol can be observed by Konelab 30.This is an expression of the reaction rate and thereby the enzymeactivity.

Reaction Conditions

Reaction pH 7.15 Temperature 37° C. Reaction Time 180 sec DetectionWavelength 405 nm Measuring Time 120 sec

Unit definition: Bacillus stearothermophilus amylase (BS-amylase)activity is measured in KNU(S), Kilo Novo Units (sterarothermophilus),relative to an enzyme standard of a declared strength. This analyticalmethod is described in more details in EB-SM-0221.02 (incorporated byreference) available from Novozymes A/S, Denmark, on request.

Determination of FAU Activity

One Fungal Alpha-Amylase Unit (FAU) is defined as the amount of enzyme,which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6, Batch9947275) per hour based upon the following standard conditions:

Substrate Soluble starch Temperature 37° C. pH 4.7 Reaction time 7-20minutes

Determination of Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity is measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard.

The standard used is AMG 300 L (from Novozymes A/S, glucoamylasewildtype Aspergillus niger G1, also disclosed in Boel et al, (1984),EMBO J. 3 (5), p. 1097-1102) and WO 92/00381). The neutral alpha-amylasein this AMG falls after storage at room temperature for 3 weeks fromapprox. 1 FAU/mL to below 0.05 FAU/mL.

The acid alpha-amylase activity in this AMG standard is determined inaccordance with the following description. In this method, 1 AFAU isdefined as the amount of enzyme, which degrades 5.260 mg starch drymatter per hour under standard conditions.

Iodine forms a blue complex with starch but not with its degradationproducts. The intensity of colour is therefore directly proportional tothe concentration of starch. Amylase activity is determined usingreverse colorimetry as a reduction in the concentration of starch underspecified analytic conditions.

Alpha-amylase _Starch + Iodine _→ _Dextrins + Oligosaccharides 40° C.,pH 2.5 _Blue/violet _t = 23 sec. _Decoloration

Standard Conditions/Reaction Conditions: (Per Minute)

_Substrate: _Starch, approx. 0.17 g/L Buffer: Citate, approx. 0.03MIodine (I₂): 0.03 g/L CaCl₂: 1.85 mM pH: 2.50 ± 0.05 Incubationtemperature: 40° C. _Reaction time: _23 seconds _Wavelength: _lambda =590 nm Enzyme concentration: 0.025 AFAU/mL Enzyme working range:0.01-0.04 AFAU/mL

If further details are preferred these can be found in EB-SM-0259.02/01available on request from Novozymes A/S, and incorporated by reference.

Determination of Pullulanase Activity (NPUN)

Endo-pullulanase activity in NPUN is measured relative to a Novozymespullulanase standard. One pullulanase unit (NPUN) is defined as theamount of enzyme that releases 1 micro mol glucose per minute under thestandard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C., 20minutes). The activity is measured in NPUN/ml using red pullulan.

1 mL diluted sample or standard is incubated at 40° C. for 2 minutes.0.5 mL 2% red pullulan, 0.5 M KCl, 50 mM citric acid, pH 5 are added andmixed. The tubes are incubated at 40° C. for 20 minutes and stopped byadding 2.5 ml 80% ethanol. The tubes are left standing at roomtemperature for 10-60 minutes followed by centrifugation 10 minutes at4000 rpm. OD of the supernatants is then measured at 510 nm and theactivity calculated using a standard curve.

EXAMPLES Example Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase (Bacillus stearothermophilusalpha-amylase with the mutations I181*+G182*+N193F truncated to 491amino acids (SEQ ID NO: 1 herein for numbering)) and alpha-amylasevariants thereof was determined by incubating the referencealpha-amylase and variants at pH 4.5 and 5.5 and temperatures of 75° C.and 85° C. with 0.12 mM CaCl₂ followed by residual activitydetermination using the EnzChek® substrate (EnzChek® Ultra Amylase assaykit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 0.5and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mMacetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.0). Twenty microlitersenzyme sample was transferred to 48-well PCR MTP and 180 microlitersstability buffer (150 mM acetate, 150 mM MES, 0.01% Triton X100, 0.12 mMCaCl₂, pH 4.5 or 5.5) was added to each well and mixed. The assay wasperformed using two concentrations of enzyme in duplicates. Beforeincubation at 75° C. or 85° C., 20 microliters was withdrawn and storedon ice as control samples. Incubation was performed in a PCR machine at75° C. and 85° C. After incubation samples were diluted to 15 ng/mL inresidual activity buffer (100 mM Acetate, 0.01% Triton X100, 0.12 mMCaCl₂, pH 5.5) and 25 microliters diluted enzyme was transferred toblack 384-MTP. Residual activity was determined using the EnzCheksubstrate by adding 25 microliters substrate solution (100micrograms/ml) to each well. Fluorescence was determined every minutefor 15 minutes using excitation filter at 485-P nm and emission filterat 555 nm (fluorescence reader is Polarstar, BMG). The residual activitywas normalized to control samples for each setup.

Assuming logarithmic decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined for thereference alpha-amylase and variant thereof as shown in Table 1.

TABLE 1 T½ (min) T½ (min) T½ (min) (pH 4.5, 75° C., (pH 4.5, 85° C., (pH5.5, 85° C., Mutations 0.12 mM CaCl₂) 0.12 mM CaCl₂) 0.12 mM CaCl₂)Reference Alpha-Amylase A 21 4 111 Reference Alpha-Amylase A with 32 6301 the substitution V59A Reference Alpha-Amylase A with 28 5 230 thesubstitution V59E Reference Alpha-Amylase A with 28 5 210 thesubstitution V59I Reference Alpha-Amylase A with 30 6 250 thesubstitution V59Q Reference Alpha-Amylase A with 149 22 ND thesubstitutions V59A + Q89R + G112D + E129V + K177L + R179E + K220P +N224L + Q254S Reference Alpha-Amylase A with >180 28 ND thesubstitutions V59A + Q89R + E129V + K177L + R179E + H208Y + K220P +N224L + Q254S Reference Alpha-Amylase A with 112 16 ND the substitutionsV59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E +D281N Reference Alpha-Amylase A with 168 21 ND the substitutions V59A +Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + I270L ReferenceAlpha-Amylase A with >180 24 ND the substitutions V59A + Q89R + E129V +K177L + R179E + K220P + N224L + Q254S + H274K Reference Alpha-Amylase Awith 91 15 ND the substitutions V59A + Q89R + E129V + K177L + R179E +K220P + N224L + Q254S + Y276F Reference Alpha-Amylase A with 141 41 NDthe substitutions V59A + E129V + R157Y + K177L + R179E + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 62 ND thesubstitutions V59A + E129V + K177L + R179E + H208Y + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 49 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S Reference Alpha-Amylase A with >180 53 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K ReferenceAlpha-Amylase A with >180 57 ND the substitutions V59A + E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + Y276F Reference Alpha-Amylase Awith >180 37 ND the substitutions V59A + E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + D281N Reference Alpha-Amylase A with >180 51 NDthe substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + M284T Reference Alpha-Amylase A with >180 45 ND thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + G416V Reference Alpha-Amylase A with 143 21 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + Q254SReference Alpha-Amylase A with >180 22 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + Q254S + M284T ReferenceAlpha-Amylase A with >180 38 ND the substitutions A91L + M96I + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 57 11 402 the substitutions E129V + K177L + R179E ReferenceAlpha-Amylase A with 174 44 >480 the substitutions E129V + K177L +R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + Y276F + L427M Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + M284T Reference Alpha-Amylase A with 177 36 >480the substitutions E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + N376* + I377* Reference Alpha-Amylase A with 94 13 >480 thesubstitutions E129V + K177L + R179E + K220P + N224L + Q254S ReferenceAlpha-Amylase A with 129 24 >480 the substitutions E129V + K177L +R179E + K220P + N224L + Q254S + M284T Reference Alpha-Amylase A with 14830 >480 the substitutions E129V + K177L + R179E + S242Q ReferenceAlpha-Amylase A with 78 9 >480 the substitutions E129V + K177L + R179VReference Alpha-Amylase A with 178 31 >480 the substitutions E129V +K177L + R179V + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 66 17 >480 the substitutions K220P + N224L + S242Q + Q254SReference Alpha-Amylase A with 30 6 159 the substitutions K220P +N224L + Q254S Reference Alpha-Amylase A with 35 7 278 the substitutionM284T Reference Alpha-Amylase A with 59 13 ND the substitutions M284V NDnot determined

The results demonstrate that the alpha-amylase variants have asignificantly greater half-life and stability than the referencealpha-amylase.

Example 2 Preparation of Protease Variants and Test of ThermostabilityStrains and Plasmids

E. coli DH12S (available from Gibco BRL) was used for yeast plasmidrescue. pJTP000 is a S. cerevisiae and E. coli shuttle vector under thecontrol of TPI promoter, constructed from pJC039 described in WO01/92502, in which the Thermoascus aurantiacus M35 protease gene (WO03/048353) has been inserted.

Saccharomyces cerevisiae YNG318 competent cells: MATa Dpep4[cir+]ura3-52, leu2-D2, his 4-539 was used for protease variants expression.It is described in J. Biol. Chem. 272 (15), pp 9720-9727, 1997.

Media and Substrates

10× Basal solution: Yeast nitrogen base w/o amino acids (DIFCO) 66.8g/l, succinate 100 g/l, NaOH 60 g/l.

SC-glucose: 20% glucose (i.e., a final concentration of 2%=2 g/100 ml))100 ml/l, 5% threonine 4 ml/l, 1% tryptophan10 ml/l, 20% casamino acids25 ml/l, 10× basal solution 100 ml/l. The solution is sterilized using afilter of a pore size of 0.20 micrometer. Agar (2%) and H₂O (approx. 761ml) is autoclaved together, and the separately sterilized SC-glucosesolution is added to the agar solution.

YPD: Bacto peptone 20 g/l, yeast extract 10 g/I, 20% glucose 100 ml/l.

YPD+Zn: YPD+0.25 mM ZnSO₄

PEG/LiAc solution: 40% PEG4000 50 ml, 5 M Lithium Acetate 1 ml.

96 well Zein micro titre plate:

Each well contains 200 microL of 0.05-0.1% of zein (Sigma), 0.25 mMZnSO₄ and 1% of agar in 20 mM sodium acetate buffer, pH 4.5.

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al. (1989) Molecular cloning: A laboratory manual, ColdSpring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R, and Cutting, S. M. (Eds.),

Yeast Transformation

Yeast transformation was performed using the lithium acetate method. 0.5microL of vector (digested by restriction endnucleases) and 1 microL ofPCR fragments is mixed. The DNA mixture, 100 microL of YNG318 competentcells, and 10 microL of YEAST MAKER carrier DNA (Clontech) is added to a12 ml polypropylene tube (Falcon 2059). Add 0.6 ml PEG/LiAc solution andmix gently. Incubate for 30 min at 30° C., and 200 rpm followed by 30min at 42° C. (heat shock). Transfer to an eppendorf tube and centrifugefor 5 sec. Remove the supernatant and resolve in 3 ml of YPD. Incubatethe cell suspension for 45 min at 200 rpm at 30° C. Pour the suspensionto SC-glucose plates and incubate 30° C. for 3 days to grow colonies.Yeast total DNA are extracted by Zymoprep Yeast Plasmid Miniprep Kit(ZYMO research).

DNA Sequencing

E. coli transformation for DNA sequencing was carried out byelectroporation (BIO-RAD Gene Pulser). DNA Plasmids were prepared byalkaline method (Molecular Cloning, Cold Spring Harbor) or with theQiagen® Plasmid Kit. DNA fragments were recovered from agarose gel bythe Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNAEngine. The ABI PRISM™ 310 Genetic Analyzer was used for determinationof all DNA sequences.

Construction of Protease Expression Vector

The Themoascus M35 protease gene was amplified with the primer pair ProtF (SEQ ID NO: 15 herein) and Prot R (SEQ ID NO: 16 herein). Theresulting PCR fragments were introduced into S. cerevisiae YNG318together with the pJC039 vector (described in WO2001/92502) digestedwith restriction enzymes to remove the Humicola insolens cutinase gene.

The Plasmid in yeast clones on SC-glucose plates was recovered toconfirm the internal sequence and termed as pJTP001.

Construction of Yeast Library and Site-Directed Variants

Library in yeast and site-directed variants were constructed by SOE PCRmethod (Splicing by Overlap Extension, see “PCR: A practical approach”,p. 207-209, Oxford University press, eds. McPherson, Quirke, Taylor),followed by yeast in vivo recombination.

General Primers for Amplification and Sequencing

The primers AM34 (SEQ ID NO: 17 herein) and AM35 (SEQ ID NO: 18 herein)were used to make DNA fragments containing any mutated fragments by theSOE method together with degenerated primers (AM34+ Reverse primer andAM35+ forward primer) or just to amplify a whole protease gene(AM34+AM35).

PCR reaction system: Conditions: 48.5 microL H₂O 1 94° C. 2 min 2 beadspuRe Taq Ready-To-Go PCR 2 94° C. 30 sec (Amersham Biosciences) 3 55° C.30 sec 0.5 micro L X 2 100 pmole/microL of primers 4 72° C. 90 sec 0.5microL template DNA 2-4 25 cycles 5 72° C. 10 min

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit. The resulting purified fragments were mixed with thevector digest. The mixed solution was introduced into Saccharomycescerevisiae to construct libraries or site-directed variants by in vivorecombination.

Relative Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate containing YPD+Zn medium and cultivated at 28° C. for 3days. The culture supernatants were applied to a 96-well zein microtiter plate and incubated at at least 2 temperatures (ex. 60° C. and 65°C., 70° C. and 75° C., 70° C. and 80° C.) for more than 4 hours orovernight. The turbidity of zein in the plate was measured as A630 andthe relative activity (higher/lower temperatures) was determined as anindicator of thermoactivity improvement. The clones with higher relativeactivity than the parental variant were selected and the sequence wasdetermined.

Remaining Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate and cultivated at 28° C. for 3 days. Protease activity wasmeasured at 65° C. using azo-casein (Megazyme) after incubating theculture supernatant in 20 mM sodium acetate buffer, pH 4.5, for 10 minat a certain temperature (80° C. or 84° C. with 4° C. as a reference) todetermine the remaining activity. The clones with higher remainingactivity than the parental variant were selected and the sequence wasdetermined.

Azo-Casein Assay

20 microL of samples were mixed with 150 microL of substrate solution (4ml of 12.5% azo-casein in ethanol in 96 ml of 20 mM sodium acetate, pH4.5, containing 0.01% triton-100 and 0.25 mM ZnSO₄) and incubated for 4hours or longer.

After adding 20 microL/well of 100% trichloroacetic acid (TCA) solution,the plate was centrifuge and 100 microL of supernatants were pipette outto measure A440.

Expression of Protease Variants in Aspergillus oryzae

The constructs comprising the protease variant genes were used toconstruct expression vectors for Aspergillus. The Aspergillus expressionvectors consist of an expression cassette based on the Aspergillus nigerneutral amylase II promoter fused to the Aspergillus nidulans triosephosphate isomerase non translated leader sequence (Pna2/tpi) and theAspergillus niger amyloglycosidase terminator (Tamg). Also present onthe plasmid was the Aspergillus selective marker amdS from Aspergillusnidulans enabling growth on acetamide as sole nitrogen source. Theexpression plasmids for protease variants were transformed intoAspergillus as described in Lassen et al. (2001), Appl. Environ.Microbiol, 67, 4701-4707. For each of the constructs 10-20 strains wereisolated, purified and cultivated in shake flasks.

Purification of Expressed Variants

-   1. Adjust pH of the 0.22 μm filtered fermentation sample to 4.0.-   2. Put the sample on an ice bath with magnetic stirring. Add    (NH4)2SO4 in small aliquots (corresponding to approx. 2.0-2.2 M    (NH4)2SO4 not taking the volume increase into account when adding    the compound).-   3. After the final addition of (NH4)2SO4, incubate the sample on the    ice bath with gentle magnetic stirring for min. 45 min.-   4. Centrifugation: Hitachi himac CR20G High-Speed Refrigerated    Centrifuge equipped with R20A2 rotor head, 5° C., 20,000 rpm, 30    min.-   5. Dissolve the formed precipitate in 200 ml 50 mM Na-acetate pH    4.0.-   6. Filter the sample by vacuum suction using a 0.22 μm PES PLUS    membrane (IWAKI).-   7. Desalt/buffer-exchange the sample to 50 mM Na-acetate pH 4.0    using ultrafiltration (Vivacell 250 from Vivascience equipped with 5    kDa MWCO PES membrane) overnight in a cold room. Dilute the    retentate sample to 200 ml using 50 mM Na-acetate pH 4.0. The    conductivity of sample is preferably less than 5 mS/cm.-   8. Load the sample onto a cation-exchange column equilibrated with    50 mM Na-acetate pH 4.0. Wash unbound sample out of the column using    3 column volumes of binding buffer (50 mM Na-acetate pH 4.0), and    elute the sample using a linear gradient, 0-100% elution buffer (50    mM Na-acetate+1 M NaCl pH 4.0) in 10 column volumes.-   9. The collected fractions are assayed by an endo-protease assay    (cf. below) followed by standard SDS-PAGE (reducing conditions) on    selected fractions. Fractions are pooled based on the endo-protease    assay and SDS-PAGE.

Endo-Protease Assay

-   1. Protazyme OL tablet/5 ml 250 mM Na-acetate pH 5.0 is dissolved by    magnetic stirring (substrate: endo-protease Protazyme AK tablet from    Megazyme—cat. #PRAK 11/08).-   2. With stirring, 250 microL of substrate solution is transferred to    a 1.5 ml Eppendorf tube.-   3. 25 microL of sample is added to each tube (blank is sample    buffer).-   4. The tubes are incubated on a Thermomixer with shaking (1000 rpm)    at 50° C. for 15 minutes.-   5. 250 microL of 1 M NaOH is added to each tube, followed by    vortexing.-   6. Centrifugation for 3 min. at 16,100×G and 25° C.-   7. 200 microL of the supernatant is transferred to a MTP, and the    absorbance at 590 nm is recorded.

Results

TABLE 2 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2 herein. Relative activity VariantSubstitution(s) 65° C./60° C. WT None 31% JTP004 S87P 45% JTP005 A112P43% JTP008 R2P 71% JTP009 D79K 69% JTP010 D79L 75% JTP011 D79M 73%JTP012 D79L/S87P 86% JTP013 D79L/S87P/A112P 90% JTP014 D79L/S87P/A112P88% JTP016 A73C 52% JTP019 A126V 69% JTP021 M152R 59%

TABLE 3 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2 herein. Relative activity Substitution(s)70° C./ 75° C./ 75° C./ Variant and/or deletion (S) 65° C. 65° C. 70° C.WT None 59% 17% JTP036 D79L/S87P/D142L 73% 73% JTP040 T54R/D79L/S87P 71%JTP042 Q53K/D79L/S87P/I173V 108%  JTP043 Q53R/D79L/S87P 80% JTP045S41R/D79L/S87P 82% JTP046 D79L/S87P/Q158W 96% JTP047 D79L/S87P/S157K 85%JTP048 D79L/S87P/D104R 88% JTP050 D79L/S87P/A112P/D142L 88% JTP051S41R/D79L/S87P/A112P/D142L 102% JTP052 D79L/S87P/A112P/D142L/S157K 111%JTP053 S41R/D79L/S87P/A112P/D142L/ 113% S157K JTP054 ΔS5/D79L/S87P  92%JTP055 ΔG8/D79L/S87P  95% JTP059 C6R/D79L/S87P  92% JTP061T46R/D79L/S87P 111% JTP063 S49R/D79L/S87P  94% JTP064 D79L/S87P/N88R 92% JTP068 D79L/S87P/T114P  99% JTP069 D79L/S87P/S115R 103% JTP071D79L/S87P/T116V 105% JTP072 N26R/D79L/S87P 92% JTP077A27K/D79L/S87P/A112P/D142L 106%  JTP078 A27V/D79L/S87P/A112P/D142L 100% JTP079 A27G/D79L/S87P/A112P/D142L 104% 

TABLE 4 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2 herein. Remaining Substitution(s)Relative activity activity Variant and/or deletion(s) 75° C./65° C. 80°C. 84° C. JTP082 ΔS5/D79L/S87P/A112P/D142L 129% 53% JTP083T46R/D79L/S87P/A112P/D142L 126% JTP088 Y43F/D79L/S87P/A112 P/D142L 119%JTP090 D79L/S87P/A112P/T124L/D142L 141% JTP091D79L/S87P/A112P/T124V/D142L 154% 43% JTP092ΔS5/N26R/D79L/S87P/A112P/D142L 60% JTP095N26R/T46R/D79L/S87P/A112P/D142L 62% JTP096 T46R/D79L/S87P/T116V/D142L67% JTP099 D79L/P81R/S87P/A112P/D142L 80% JTP101A27K/D79L/S87P/A112P/T124V/D142L 81% JTP116D79L/Y82F/S87P/A112P/T124V/D142L 59% JTP117D79L/Y82F/S87P/A112P/T124V/D142L 94% JTP127D79L/S87P/A112P/T124V/A126V/D142L 53%

TABLE 5 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2 herein. Relative activity VariantSubstitutions 75° C./70° C. 80° C./70° C. 85° C./70° C. JTP050 D79L S87PA112P D142L 55% 23%  9% JTP134 D79L Y82F S87P A112P D142L 40% JTP135S38T D79L S87P A112P A126V D142L 62% JTP136 D79L Y82F S87P A112P A126VD142L 59% JTP137 A27K D79L S87P A112P A126V D142L 54% JTP140 D79L S87PN98C A112P G135C D142L 81% JTP141 D79L S87P A112P D142L T141C M161C 68%JTP143 S36P D79L S87P A112P D142L 69% JTP144 A37P D79L S87P A112P D142L57% JTP145 S49P D79L S87P A112P D142L 82% 59% JTP146 S50P D79L S87PA112P D142L 83% 63% JTP148 D79L S87P D104P A112P D142L 76% 64% JTP161D79L Y82F S87G A112P D142L 30% 12% JTP180 S70V D79L Y82F S87G Y97W A112PD142L 52% JTP181 D79L Y82F S87G Y97W D104P A112P D142L 45% JTP187 S70VD79L Y82F S87G A112P D142L 45% JTP188 D79L Y82F S87G D104P A112P D142L43% JTP189 D79L Y82F S87G A112P A126V D142L 46% JTP193 Y82F S87G S70VD79L D104P A112P D142L 15% JTP194 Y82F S87G D79L D104P A112P A126V D142L22% JTP196 A27K D79L Y82F S87G D104P A112P A126V D142L 18%

TABLE 6 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2 herein. Relative activity VariantSubstitutions 75° C./70° C. 80° C./70° C. JTP196 A27K D79L Y82F S87GD104P A112P A126V D142L 102% 55% JTP210 A27K Y82F S87G D104P A112P A126VD142L 107% 36% JTP211 A27K D79L Y82F D104P A112P A126V D142L  94% 44%JTP213 A27K Y82F D104P A112P A126V D142L 103% 37%

Example 3 Temperature Profile of Selected Variants Using PurifiedEnzymes

Selected variants showing good thermo-stability were purified and thepurified enzymes were used in a zein-BCA assay as described below. Theremaining protease activity was determined at 60° C. after incubation ofthe enzyme at elevated temperatures as indicated for 60 min,

Zein-BCA Assay:

Zein-BCA assay was performed to detect soluble protein quantificationreleased from zein by variant proteases at various temperatures,

Protocol:

-   1) Mix 10 microliters of 10 micrograms/ml enzyme solutions and 100    ul of 0.025% zein solution in a micro titer plate (MTP).-   2) Incubate at various temperatures for 60 min.-   3) Add 10 microliters of 100% trichloroacetic acid (TCA) solution,-   4) Centrifuge MTP at 3500 rpm for 5 min.-   5) Take out 15 microliters to a new MTP containing 100 microliters    of BCA assay solution (Pierce Cat#:23225, BCA Protein Assay Kit).-   6) Incubate for 30 min. at 60° C.-   7) Measure A562.

The results are shown in Table 7. All of the tested variants showed animproved thermo-stability as compared to the wt protease.

TABLE 7 Zein-BCA assay Sample incubated 60 min at indicated temperatures(° C.) (μg/ml Bovine serum albumin equivalent peptide released) 60° 70°75° 80° 85° 90° 95° WT/Variant C. C. C. C. C. C. C. WT 94 103 107 93 5838 JTP050 86 101 107 107 104 63 36 JTP077 82 94 104 105 99 56 31 JTP18871 83 86 93 100 75 53 JTP196 87 99 103 106 117 90 38

Example 4

Characterization of Penicillium oxalicum Glucoamylase

The Penicillium oxalicum glucoamylase is disclosed in SEQ ID NO: 14(mature) herein.

Substrate. Substrate: 1% soluble starch (Sigma S-9765) in deionizedwater

Reaction buffer: 0.1M Acetate buffer at pH 5.3

Glucose concentration determination kit: Wako glucose assay kit(LabAssay glucose, WAKO, Cat#298-65701),

Reaction condition. 20 microL soluble starch and 50 microL acetatebuffer at pH 5.3 were mixed. 30 microL enzyme solution (50 micro genzyme protein/rill) was added to a final volume of 100 microL followedby incubation at 37° C. for 15 min.

The glucose concentration was determined by Wako kits.

All the work carried out in parallel.

Temperature optimum. To assess the temperature optimum of thePenicillium oxalicum glucoamylase the “Reaction condition”-assaydescribed above was performed at 20, 30, 40, 50, 60, 70, 80, 85, 90 and95° C. The results are shown in Table 8.

TABLE 8 Temperature optimum Temperature (° C.) 20 30 40 50 60 70 80 8590 95 Relative activity (%) 63.6 71.7 86.4 99.4 94.6 100.0 92.9 92.582.7 82.8

From the results it can be seen that the optimal temperature forPenicillium oxalicum glucoamylase at the given conditions is between 50°C. and 70° C. and the glucoamylase maintains more than 80% activity at95° C.

Heat stability. To assess the heat stability of the Penicillium oxalicumglucoamylase the Reaction condition assay was modifed in that the theenzyme solution and acetate buffer was preincubated for 15 min at 20,30, 40, 50, 60, 70, 75, 80, 85, 90 and 95° C. Following the incubation20 microL of starch was added to the solution and the assay wasperformed as described above.

The results are shown in Table 9.

TABLE 9 Heat stability Temperature (° C.) 20 30 40 50 60 70 80 85 90 95Relative activity (%) 91.0 92.9 88.1 100.0 96.9 86.0 34.8 36.0 34.2 34.8

From the results it can be seen that Penicillium oxalicum glucoamylaseis stable up to 70° C. after preincubation for 15 min in that itmaintains more than 80% activity.

pH optimum. To assess the pH optimum of the Penicillium oxalicumglucoamylase the Reaction condition assay described above was performedat pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0.Instead of using the acetate buffer described in the Reaction conditionassay the following buffer was used 100 mM Succinic acid, HEPES, CHES,CAPSO, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 2.0,3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl orNaOH.

The results are shown in Table 10.

TABLE 10 pH optimum pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.0 11.0Relative activity (%) 71.4 78.6 77.0 91.2 84.2 100.0 55.5 66.7 30.9 17.815.9 16.1

From the results it can be seen that Penicillium oxalicum glucoamylaseat the given conditions has the highest activity at pH 5.0. ThePenicillium oxalicum glucoamylase is active in a broad pH range in theit maintains more than 50° /h activity from pH 2 to 7.

pH stability. To assess the heat stability of the Penicillium oxalicumglucoamylase the Reaction condition assay was modifed in that the enzymesolution (50 micro g/mL) was preincubated for 20 hours in buffers withpH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0 usingthe buffers described under pH optimum. After preincubation, 20 microLsoluble starch to a final volume of 100 microL was added to the solutionand the assay was performed as described above.

The results are shown in Table 11.

TABLE 11 pH stability pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.011.0 Relative activity (%) 17.4 98.0 98.0 103.2 100.0 93.4 71.2 90.758.7 17.4 17.0 17.2

From the results it can be seen that Penicillium oxalicum glucoamylase,is stable from pH 3 to pH 7 after preincubation for 20 hours and itdecreases its activity at pH 8.

Example 5 Thermostability of Protease Pfu.

The thermostability of the Pyrococcus furiosus protease (Pfu S)purchased from Takara Bio Inc, (Japan) was tested using the same methodsas in Example 2. It was found that the thermostability (RelativeActivity) was 110% at (80° C./70° C.) and 103% (90° C./70° C.) at pH4.5.

Example 6

Cloning of Penicillium oxalicum Strain Glucoamylase GenePreparation of Penicillium oxalicum Strain cDNA.

The cDNA was synthesized by following the instruction of 3′ RapidAmplifiction of cDNA End System (Invitrogen Corp., Carlsbad, Calif.,USA).

Cloning of Penicillium oxalicum Strain Glucoamylase Gene.

The Penicillium oxalicum glucoamylase gene was cloned using theoligonucleotide primer shown below designed to amplify the glucoamylasegene from 5′ end.

Sense primer: (SEQ ID NO: 19) 5′-ATGCGTCTCACTCTATTATCAGGTG-3′

The full length gene was amplified by PCR with Sense primer and AUAP(supplied by 3′ Rapid Amplifiction of cDNA End System) by using PlatinumHIFI Taq DNA polymerase (Invitrogen Corp., Carlsbad, Calif., USA). Theamplification reaction was composed of 5 μl of 10× PCR buffer, 2 μl of25 mM MgCl₂, 1 μl of 10 mM dNTP, 1 μl of 10 uM Sense primer, 1 μl of 10uM AUAP, 2 μl of the first strand cDNA, 0.5 μl of HIFI Taq, and 37.5 μlof deionized water. The PCR program was: 94° C., 3 mins; 10 cycles of94° C. for 40 secs, 60° C. 40 secs with 1° C. decrease per cycle, 68° C.for 2 min; 25 cycles of 94° C. for 40 secs, 50° C. for 40 secs, 68° C.for 2 min; final extension at 68° C. for 10 mins.

The obtained PCR fragment was cloned into pGEM-T vector (PromegaCorporation, Madison, Wis., USA) using a pGEM-T Vector System (PromegaCorporation, Madison, Wis., USA) to generate plasmid AMG 1. Theglucoamylase gene inserted in the plasmid AMG 1 was sequencingconfirmed. E. coli strain TOP10 containing plasmid AMG 1 (designatedNN059173), was deposited with the Deutsche Sammlung von MikroorganismenLind Zellkulturen GmbH (DSMZ) on Nov. 23, 2009, and assigned accessionnumber as DSM 23123.

Example 7

Expression of Cloned Penicillium oxalicum Glucoamylase

The Penicillium oxalicum glucoamylase gene was re-cloned from theplasmid AMG 1 into an Aspergillus expression vector by PCR using twocloning primer F and primer R shown below, which were designed based onthe known sequence and added tags for direct cloning by lN-FUSION™strategy.

Primer F: (SEQ ID NO: 20) 5′ ACACAACTGGGGATCCACCATGCGTCTCACTCTATTATCPrimer R: (SEQ ID NO: 21) 5′ AGATCTCGAGAAGCTTAAAACTGCCACACGTCGTTGG

A PCR reaction was performed with plasmid AMG 1 in order to amplify thefull-length gene. The PCR reaction was composed of 40 μg of the plasmidAMG 1 DNA, 1 μl of each primer (100 μM); 12.5 μl of 2× ExtensorHi-Fidelity master mix (Extensor Hi-Fidelity Master Mix, ABgene, UnitedKingdom), and 9.5 μl of PCR-grade water. The PCR reaction was performedusing a DYAD PCR machine (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) programmed for 2 minutes at 94° C. followed by a 25 cycles of 94°C. for 15 seconds, 50° C. for 30 seconds, and 72° C. for 1 minute; andthen 10 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 1× TAE buffer where an approximately 1.9 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit (GE Healthcare, United Kingdom) according tomanufacturer's instructions. DNA corresponding to the Penicilliumoxalicum glucoamylase gene was cloned into an Aspergillus expressionvector linearized with BamHI and HindIII, using an IN-FUSION™ Dry-DownPCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) according tothe manufacturer's instructions. The linearized vector construction isas described in WO 2005/042735 A1.

A 2 μl volume of the ligation mixture was used to transform 25 μl ofFusion Blue E. coli cells (included in the IN-FUSION™ Dry-Down PCRCloning Kit). After a heat shock at 42° C. for 45 sec, and chilling onice, 250 μl of SOC medium was added, and the cells were incubated at 37°C. at 225 rpm for 90 min before being plated out on LB agar platescontaining 50 μg of ampicillin per ml, and cultivated overnight at 37°C. Selected colonies were inoculated in 3 ml of LB medium supplementedwith 50 μg of ampicillin per ml and incubated at 37° C. at 225 rpmovernight. Plasmid DNA from the selected colonies was purified usingMini JETSTAR (Genomed, Germany) according to the manufacturer'sinstructions. Penicillium oxalicum glucoamylase gene sequence wasverified by Sanger sequencing before heterologous expression. One of theplasmids was selected for further expression, and was named XYZXYZ1471-4.

Protoplasts of Aspergillus niger MBin118 were prepared as described inWO 95/02043. One hundred μl of protoplast suspension were mixed with 2.5μg of the XYZ1471-4 plasmid and 250 microliters of 60% PEG 4000(Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂,and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The mixture wasincubated at 37° C. for 30 minutes and the protoplasts were mixed with6% low melting agarose (Biowhittaker Molecular Applications) in COVEsucrose (Cove, 1996, Biochim. Biophys, Acta 133:51-56) (1M) platessupplemented with 10 mM acetamide and 15 mM CsCl and added as a toplayer on COVE sucrose (1M) plates supplemented with 10 mM acetamide and15 mM CsCl for transformants selection (4 ml topagar per plate). Afterincubation for 5 days at 37° C. spores of sixteen transformants werepicked up and seed on 750 μl YP-2% Maltose medium in 96 deepwell MTplates. After 5 days of stationary cultivation at 30° C., 10 μl of theculture-broth from each well was analyzed on a SDS-PAGE (Sodium dodecylsulfate-polyacrylamide gel electrophoresis) gel, Griton XT Precast gel(BioRad, CA, USA) in order to identify the best transformants based onthe ability to produce large amount of glucoamylase. A selectedtransformant was identified on the original transformation plate and waspreserved as spores in a 20% glycerol stock and stored frozen (−80° C.).

Cultivation. The selected transformant was inoculated in 100 ml of MLCmedia and cultivated at 30° C. for 2 days in 500 ml shake flasks on arotary shaker. 3 ml of the culture broth was inoculated to 100 ml ofM410 medium and cultivated at 30° C. for 3 days. The culture broth wascentrifugated and the supernatant was filtrated using 0.2 μm membranefilters.

Alpha-cyclodextrin affinity gel. Ten grams of Epoxy-activated Sepharose6B (GE Healthcare, Chalfont St, Giles, U.K) powder was suspended in andwashed with distilled water on a sintered glass filter. The gel wassuspended in coupling solution (100 ml of 12.5 mg/ml alpha-cyclodextrin,0.5 M NaOH) and incubated at room temperature for one day with gentleshaking. The gel was washed with distilled water on a sintered glassfilter, suspended in 100 ml of 1 M ethanolamine, pH 10, and incubated at50° C. for 4 hours for blocking. The gel was then washed several timesusing 50 mM Tris-HCl, pH 8 and 50 mM NaOAc, pH 4.0 alternatively. Thegel was finally packed in a 35-40 ml column using equilibration buffer(50 mM NaOAc, 150 mM NaCl, pH 4.5).

Purification of glucoamylase from culture broth. Culture broth fromfermentation of A. niger MBin118 harboring the glucoamylase gene wasfiltrated through a 0.22 μm RES filter, and applied on aalpha-cyclodextrin affinity gel column previously equilibrated in 50 mMNaOAc, 150 mM NaCl, pH 4.5 buffer. Unbound material was washed off thecolumn with equilibration buffer and the glucoamylase was eluted usingthe same buffer containing 10 mM beta-cyclodextrin over 3 columnvolumes.

The glucoamylase activity of the eluent was checked to see, if theglucoamylase had bound to the alpha-cyclodextrin affinity gel. Thepurified glucoamylase sample was then dialyzed against 20 mM NaOAc, pH5.0. The purity was finally checked by SDS-PAGE, and only a single bandwas found.

Example 8

Construction and Expression of a Site-Directed Variant of Penicilliumoxalicum Glucoamylase (PEON)

Two PCR reactions were performed with plasmid XYZ1471-4, described inExample 9, using primers K79V F and K79VR shown below, which weredesigned to substitute lysine K at position 79 from the mature seequenceto varin V and primers F-NP003940 and R-NP003940 shown below, which weredesigned based on the known sequence and added tags for direct cloningby IN-FUSION™ strategy.

Primer K79V F 18mer (SEQ ID NO: 22) GCAGTCTTTCCAATTGACPrimer K79V R 18mer (SEQ ID NO: 23) AATTGGAAAGACTGCCCGPrimer F-NP003940: (SEQ ID NO: 24) 5′ACACAACTGGGGATCCACCATGCGTCTCACTCTATTATC Primer R-NP003940:(SEQ ID NO: 25) 5′ AGATCTCGAGAAGCTTAAAACTGCCACACGTCGTTGG

The PCR was performed using a PTC-200 DNA Engine under the conditionsdescribed below.

PCR reaction system: Conditions: 48.5 micro L H2O 1 94° C. 2 min 2 beadspuRe Taq Ready-To-Go PCR 2 94° C. 30 sec Beads (Amersham bioscineces) 355° C. 30 sec O.Smicro L X 2100 pmole/micro L Primers 4 72° C. 90 sec(K79V F + Primer R-NP003940, K79V R + 2-4 25 cycles Primer F-NP003940) 572° C. 10 min 0.5 micro L Template DNA

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit according to the manufacturer's instruction. Theresulting purified two fragments were cloned into an Aspergillusexpression vector linearized with BamHI and HindIII, using an IN-FUSION™Dry-Down PCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA)according to the manufacturer's instructions. The linearized vectorconstruction is as described in WO 2005/042735 A1.

The ligation mixture was used to transform E. coli DH5α cells (TOYOBO).Selected colonies were inoculated in 3 ml of LB medium supplemented with50 μg of ampicillin per ml and incubated at 37° C. at 225 rpm overnight.Plasmid DNA from the selected colonies was purified using Qiagen plasmidmini kit (Qiagen) according to the manufacturer's instructions. Thesequence of Penicillium oxalicum glucoamylase site-directed variant genesequence was verified before heterologous expression and one of theplasmids was selected for further expression, and was named pPoPE001.

Protoplasts of Aspergillus niger MBin118 were prepared as described inWO 95/02043. One hundred microliters of protoplast suspension were mixedwith 2.5 μg of the pPoPE001 plasmid and 250 microliters of 60% PEG 4000(Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂,and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The mixture wasincubated at 37° C. for 30 minutes and the protoplasts were mixed with1% agarose L (Nippon Gene) in COVE sucrose (Cove, 1996, Biochim.Biophys. Acta 133:51-56) supplemented with 10 mM acetamide and 15 mMCsCl and added as a top layer on COVE sucrose plates supplemented with10 mM acetamide and 15 mM CsCl for transformants selection (4 ml topagarper plate). After incubation for 5 days at 37° C. spores of sixteentransformants were picked up and seed on 750 μl YP-2% Maltose medium in96 deepwell MT plates. After 5 days of stationary cultivation at 30° C.,10 μl of the culture-broth from each well was analyzed on a SDS-PAGE gelin order to identify the best transformants based on the ability toproduce large amount of the glucoamylase.

Example 9 Purification of Site-Directed Po AMG Variant PE001

The selected transformant of the variant and the strain expressing thewild type Penicillium oxalicum glucoamylase described in Example 8 wascultivated in 100 ml of YP-2% maltose medium and the culture wasfiltrated through a 0.22 μm RES filter, and applied on aalpha-cyclodextrin affinity gel column previously equilibrated in 50 mMNaOAc, 150 mM NaCl, pH 4.5 buffer. Unbound materias was washed off thecolumn with equilibration buffer and the glucoamylase was eluted usingthe same buffer containing 10 mM beta-cyclodextrin over 3 columnvolumes.

The glucoamylase activity of the eluent was checked to see, if theglucoamylase had bound to the alpha-cyclodextrin affinity gel. Thepurified glucoamylase samples were then dialyzed against 20 mM NaOAc, pH5.0.

Example 10 Characterization of PE001 Protease Stability

40 μl enzyme solutions (1 mg/ml) in 50 mM sodium acetate buffer, pH 4.5,was mixed with 1/10 volume of 1 mg/ml protease solutions such asaspergillopepsinl described in Biochem J. 1975 April; 147(1): 45-53 orthe commercially availble product from Sigma and aorsin described inBiochemical journal [0264-6021] lchishima, 2003, 371(2): 541 andincubated at 4 or 32° C. overnight. As a control experiment, H₂O wasadded to the sample instead of proteases. The samples were loaded onSDS-PAGE to see if the glucoamylases are cleaved by proteases.

In SDS-PAGE, PE001 only showed one band corresponding to the intactmolecule, while the wild type glucoamylase was degraded by proteases andshowed a band at lower molecular size at 60 kCa.

TABLE 12 The result of SDS-PAGE after protease treatment Wild typeglucoamylase PE001 Protease aspergillopepsin I aorsin aspergillopepsin Iaorsin control Incubation temperature (° C.) 4 32 4 32 4 32 4 32 4intact glucoamylase 100% 90% 40% 10% 100% 100% 100% 100% 100% (ca. 70kDa) cleaved glucoamylase N.D. 10% 60% 90% N.D. N.D. N.D. N.D. N.D. (ca.60 kDa) N.D.: not detected.

Example 11 Less Cleavage During Cultivation

Aspergillus transformant of the variant (PE001) and the wild typePenicillium oxalicum glucoamylase were cultivated in 6-well MT platescontaining 4× diluted YR-2% maltose medium supplemented with 10 mMsodium acetate buffer, pH4.5, at 32° C. for 1 week.

The culture supernatants were loaded on SDS-PAGE.

TABLE 13 The result of SDS-PAGE of the culture supernatants Wild typeglucoamylase PE001 intact glucoamylase 90% 100% (ca. 70 kDa) cleavedglucoamylase 10% N.D. (ca. 60 kDa) N.D.: not detected.

The wild type glucoamylase was cleaved by host proteasaes duringfermentation, while the variant yielded only intact molecule.

Example 12 Glucoamylase Activity of Variant PE001 Compared to Parent

The glucoamylase activity measures as AGU as described above was checkedfor the purified enzymes of the wild type Penicillium oxalicum and thevariant glucoamylase.

The Glucoamylase Unit (AGU) was defined as the amount of enzyme, whichhydrolyzes 1 micromole maltose per minute under the standard conditions(37° C., pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M,reaction time 6 minutes).

TABLE 14 Relative specific activity AGU/mg Penicillium oxalicum wt 100%Penicillium oxalicum PE001 102%

Example 13 Purification of Glucoamylase Variants Having IncreasedThermostability

The variants showing increased thermostability may be constructed andexpressed similar to the procedure described in Example 8. All variantswere derived from the PE001. After expression in YPM medium, variantscomprising the T65A or Q327F (SEQ ID NO: 14 numbering) substitution wasmicro-purified as follows:

Mycelium was removed by filtration through a 0.22 μm filter, 50 μlcolumn material (alpha-cyclodextrin coupled to Mini-Leakdivinylsulfone-activated agarose medium according to manufacturersrecommendations) was added to the wells of a filter plate (Whatman,Unifilter 800 μl, 25-30 μm MBPP). The column material was equilibratedwith binding buffer (200 mM sodium acetate pH 4.5) by two times additionof 200 μl buffer, vigorous shaking for 10 min (Heidolph, Titramax 101,1000 rpm) and removal of buffer by vacuum (Whatman, UniVac 3),Subsequently, 400 μl culture supernatant and 100 μl binding buffer wasadded and the plate incubated 30 min with vigorous shaking. Unboundmaterial was removed by vacuum and the binding step was repeated.Normally 4 wells were used per variant. Three washing steps were thenperformed with 200 μl buffer of decreasing ionic strength added (50/10/5mM sodium acetate, pH 4.5), shaking for 15 min and removal of buffer byvacuum. Elution of the bound AMG was achieved by two times addition of100 μl elution buffer (250 mM sodium acetate, 0.1% alpha-cyclodextrin,pH 6.0), shaking for 15 min and collection of eluted material in amicrotiter plate by vacuum. Pooled eluates were concentrated and bufferchanged to 50 mM sodium acetate pH 4.5 using centrifugal filter unitswith 10 kDa cut-off (Millipore Microcon Ultracel YM-10). Micropurifiedsamples were stored at −18° C. until testing of thermostability.

Example 14 Protein Thermal Unfolding Analysis (TSA, Thermal Shift Assay)

Protein thermal unfolding of the T65A and Q327F variants, was monitoredusing Sypro Orange (In-vitrogen, S-6650) and was performed using areal-time PCR instrument (Applied Biosystems; Step-One-Plus).

In a 96-well plate, 25 microliter micropurified sample in 50 mM AcetatepH4.5 at approx. 100 microgram/ml was mixed (5:1) with Sypro Orange(resulting conc.=5×; stock solution from supplier=5000×). The plate wassealed with an optical PCR seal. The PCR instrument was set at ascan-rate of 76° C. pr. hr, starting at 25° C. and finishing at 96° C.

Protein thermal unfolding of the E501V+Y504T variant, was monitoredusing Sypro Orange (In-vitrogen, S-6650) and was performed using areal-time PCR instrument (Applied Biosystems; Step-One-Plus).

In a 96-well plate, 15 microliter purified sample in 50 mM Acetate pH4.5at approx. 50 microgram/ml was mixed (1:1) with Sypro Orange (resultingconc.=5×; stock solution from supplier=5000×) with or without 200 ppmAcarbose (Sigma A8980). The plate was sealed with an optical PCR seal.The PCR instrument was set at a scan-rate of 76 degrees C. pr. hr,starting at 25° C. and finishing at 96° C.

Fluorescence was monitored every 20 seconds using in-built LED bluelight for excitation and ROX-filter (610 nm, emission).

Tm-values were calculated as the maximum value of the first derivative(dF/dK) (ref.: Gregory et al., 2009, J. Biomol. Screen. 14: 700).

TABLE 15 a. Sample Tm (Deg. Celsius) + /− 0.4 PO-AMG (PE001) 80.3Variant Q327F 82.3 Variant T65A 81.9 b. Sample Tm (Deg. Celsius) + /−0.4 Acarbose: − + PO-AMG (PE001) 79.5 86.9 Variant E501V Y504T 79.5 95.2

Example 15 Thermostability Analysis by Differential Scanning Calorimetry(DSC)

Additional site specific variants having substitutions and/or deletionsat specific positions were constructed basically as described in Example8 and purified as described in Example 9.

The thermostability of the purified Po-AMG PE001 derived variants weredetermined at pH 4.0 or 4.8 (50 mM Sodium Acetate) by DifferentialScanning calorimetry (DSC) using a VP-Capillary Differential Scanningcalorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermaldenaturation temperature, Td (° C.), was taken as the top of thedenaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating enzyme solutions in selected buffers (50 mMSodium Acetate, pH 4.0 or 4.8) at a constant programmed heating rate of200 K/hr.

Sample- and reference-solutions (approximately 0.3 ml) were loaded intothe calorimeter (reference: buffer without enzyme) from storageconditions at 10° C. and thermally pre-equilibrated for 10 minutes at20° C. prior to DSC scan from 20° C. to 110° C. Denaturationtemperatures were determined with an accuracy of approximately +/−1° C.

The isolated variants and the DSC data are disclosed in Table 16 below.

TABLE 16 Po- DSC Td DSC Td AMG Mutations (° C.) @ (° C.) @ nameMutations relative to PE001 pH 4.0 pH 4.8 PE001 82.1 83.4 GA167 E501VY504T 82.1 GA481 T65A K161S 84.1 86.0 GA487 T65A Q405T 83.2 GA490 T65AQ327W 87.3 GA491 T65A Q327F 87.7 GA492 T65A Q327Y 87.3 GA493 P11F T65AQ327F 87.8 88.5 GA497 R1K D3W K5Q G7V N8S T10K P11S 87.8 88.0 T65A Q327FGA498 P2N P4S P11F T65A Q327F 88.3 88.4 GA003 P11F D26C K33C T65A Q327F83.3 84.0 GA009 P2N P4S P11F T65A Q327W E501V 88.8 Y504T GA002 R1E DSNP4G G6R G7A N8A T10D 87.5 88.2 P11D T65A Q327F GA005 P11F T65A Q327W87.4 88.0 GA008 P2N P4S P11F T65A Q327F E501V 89.4 90.2 Y504T GA010 P11FT65A Q327W E501V Y504T 89.7 GA507 T65A Q327F E501V Y504T 89.3 GA513 T65AS105P Q327W 87.0 GA514 T65A S105P Q327F 87.4 GA515 T65A Q327W S364P 87.8GA516 T65A Q327F S364P 88.0 GA517 T65A S103N Q327F 88.9 GA022 P2N P4SP11F K34Y T65A Q327F 89.7 GA023 P2N P4S P11F T65A Q327F D445N 89.9 V447SGA032 P2N P4S P11F T65A I172V Q327F 88.7 GA049 P2N P4S P11F T65A Q327FN502* 88.4 GA055 P2N P4S P11F T65A Q327F N502T 88.0 P563S K571E GA057P2N P4S P11F R31S K33V T65A 89.5 Q327F N564D K571S GA058 P2N P4S P11FT65A Q327F S377T 88.6 GA064 P2N P4S P11F T65A V325T Q327W 88.0 GA068 P2NP4S P11F T65A Q327F D445N 90.2 V447S E501V Y504T GA069 P2N P4S P11F T65AI172V Q327F 90.2 E501V Y504T GA073 P2N P4S P11F T65A Q327F S377T 90.1E501V Y504T GA074 P2N P4S P11F D26N K34Y T65A 89.1 Q327F GA076 P2N P4SP11F T65A Q327F I375A 90.2 E501V Y504T GA079 P2N P4S P11F T65A K218AK221D 90.9 Q327F E501V Y504T GA085 P2N P4S P11F T65A S103N Q327F 91.3E501V Y504T GA086 P2N P4S T10D T65A Q327F E501V 90.4 Y504T GA088 P2N P4SF12Y T65A Q327F E501V 90.4 Y504T GA097 K5A P11F T65A Q327F E501V 90.0Y504T GA101 P2N P4S T10E E18N T65A Q327F 89.9 E501V Y504T GA102 P2N T10EE18N T65A Q327F E501V 89.8 Y504T GA084 P2N P4S P11F T65A Q327F E501V90.5 Y504T T568N GA108 P2N P4S P11F T65A Q327F E501V 88.6 Y504T K524TG526A GA126 P2N P4S P11F K34Y T65A Q327F 91.8 D445N V447S E501V Y504TGA129 P2N P4S P11F R31S K33V T65A 91.7 Q327F D445N V447S E501V Y504TGA087 P2N P4S P11F D26N K34Y T65A 89.8 Q327F E501V Y504T GA091 P2N P4SP11F T65A F80* Q327F 89.9 E501V Y504T GA100 P2N P4S P11F T65A K112SQ327F 89.8 E501V Y504T GA107 P2N P4S P11F T65A Q327F E501V 90.3 Y504TT516P K524T G526A GA110 P2N P4S P11F T65A Q327F E501V 90.6 N502T Y504*

Example 16

Thermostability Analysis by Thermo-Stress Test and pNPG Assay

Starting from one of the identified substitution variants from Example10, identified as PE008, additional variants were tested by athereto-stress assay in which the supernatant from growth cultures wereassayed for glucoamylase (AMG) activity after a heat shock at 83° C. for5 min.

After the heat-shock the residual activity of the variant was measuredas well as in a non-stressed sample.

Description of Po-AMG pNPG Activity Assay:

The Penicillium oxalicum glucoamylase pNPG activity assay is aspectrometric endpoint assay where the samples are split in two andmeasured thereto-stressed and non-thermo-stressed. The data output istherefore a measurement of residual activity in the stressed samples.

Growth:

A sterile micro titer plate (MTP) was added 200 microliters rich growthmedia (FT X-14 without Dowfax) to each well. The strains of interestwere inoculated in triplicates directly from frozen stocks to the MTP.Benchmark was inoculated in 20 wells. Non-inoculated wells with mediawere used as assay blanks. The MTP was placed in a plastic boxcontaining wet tissue to prevent evaporation from the wells duringincubation. The plastic box was placed at 34° C. for 4 days.

Assay:

50 microliters supernatant was transferred to 50 microliters 0.5 M NaAcpH 4.8 to obtain correct sample pH.

50 microliters dilution was transferred to a PCR plate andthermo-stressed at 83° C. for 5 minutes in a PCR machine. The remaininghalf of the dilution was kept at RT.

20 microliters of both stressed and unstressed samples was transferredto a standard MTP, 20 microliters pNPG-substrate was added to start thereaction. The plate was incubated at RT for 1 h.

The reaction was stopped and the colour developed by adding 50microliters 0.5 M Na₂CO₃. The yellow colour was measured on a platereader (Molecular Devices) at 405 nm.

Buffers:

0.5 M NaAc pH 4.8

0.25 M NaAc pH 4.8

Substrate, 6 mM pNPG:

15 mg 4-nitrophenyl D-glucopyranoside in 10 mL 0.25 NaAc pH 4.8

Stop/Developing Solution:

0.5 M Na₂CO₃

Data Treatment:

In Excel the raw Abs405 data from both stressed and unstressed sampleswere blank subtracted with their respective blanks. The residualactivity (% res. act.=(Abs_(unstressed)(Abs_(unstressed)−Abs_(stressed)))/Abs_(unstressed)*100%) was calculatedand plotted relative to benchmark, Po-amg0008.

TABLE 17 Po- AMG % residual name Mutations activity GA008 P2N P4S P11FT65A Q327F E501V Y504T 100 GA085 P2N P4S P11F T65A S103N Q327F E501VY504T 127 GA097 K5A P11F T65A Q327F E501V Y504T 106 GA107 P2N P4S P11FT65A Q327F E501V Y504T T516P 109 K524T G526A GA130 P2N P4S P11F T65AV79A Q327F E501V Y504T 111 GA131 P2N P4S P11F T65A V79G Q327F E501VY504T 112 GA132 P2N P4S P11F T65A V79I Q327F E501V Y504T 101 GA133 P2NP4S P11F T65A V79L Q327F E501V Y504T 102 GA134 P2N P4S P11F T65A V79SQ327F E501V Y504T 104 GA150 P2N P4S P11F T65A L72V Q327F E501V Y504T 101GA155 S255N Q327F E501V Y504T 105

TABLE 18 % Po-AMG residual name Mutations activity GA008 P2N P4S P11FT65A Q327F E501V Y504T 100 GA179 P2N P4S P11F T65A E74N V79K Q327F E501V108 Y504T GA180 P2N P4S P11F T65A G220N Q327F E501V Y504T 108 GA181 P2NP4S P11F T65A Y245N Q327F E501V Y504T 102 GA184 P2N P4S P11F T65A Q253NQ327F E501V Y504T 110 GA185 P2N P4S P11F T65A D279N Q327F E501V Y504T108 GA186 P2N P4S P11F T65A Q327F S359N E501V Y504T 108 GA187 P2N P4SP11F T65A Q327F D370N E501V Y504T 102 GA192 P2N P4S P11F T65A Q327FV460S E501V Y504T 102 GA193 P2N P4S P11F T65A Q327F V460T P468T E501V102 Y504T GA195 P2N P4S P11F T65A Q327F T463N E501V Y504T 103 GA196 P2NP4S P11F T65A Q327F S465N E501V Y504T 106 GA198 P2N P4S P11F T65A Q327FT477N E501V Y504T 106

Example 17 Test for Glucoamylase Activity of Thermo-Stable VariantsAccording to the Invention

All of the above described variants disclosed in tables 16, 17, and 18have been verified for Glucoamylase activity on culture supernatantsusing the pNPG assay described in Example 16.

Example 18

Determination of Thermostablity of Endo-beta-glucanases (EG) byDifferential Scanning Calorimitry (DSC)

The thermostability of EGs was tested as described in the “Materials &Methods” section under “Determination of Td by Differential Scanningcalorimetry for Endoglucanases and Hemicellulases”

TABLE 19 Melting ID Family Donor point ° C. (DSC) Endoglucanase TL GH5Talaromyces 89 (SEQ ID NO: 9) leycettanus Endoglucanase PC GH5Penicillium 83 (SEQ ID NO: 35) capsulatum Endoglucanase TS GH5Trichophaea saccata 81 (SEQ ID NO: 36) Endoglucanase SF GH45 Sordariafimicola 75 (SEQ ID NO: 38)

Example 19 Adding Thermostable Xylanase in Liquefaction—Effect onEthanol Yield and Corn Fiber Degradation

All treatments were evaluated via 100 g small-scale liquefaction. Cornflour obtained from industrial corn ethanol plants was used for theexperiments. The dry solids (DS) of the corn flour was 85.12%,determined by Mettler-Toledo HB43 halogen moisture balance. Forliquefaction, 38.77 g corn flour and 61.23 g tap water were added toreach DS of 33% and mass of 100 g in each canister of Lab-O-Mat. The pHof the corn slurry was found to be about 5.9 without adjustment. 40%H₂SO₄ was added to each canister to adjust pH to 5.0 for liquefaction.Each canister was dosed with the appropriate amount of diluted enzyme asshown in Table 21 below. The alpha-amylase (AA369) and theendo-glucanase (TI EG) was used. Two thermostable xylanases wereevaluated as shown in Table 20. Final DS of the corn slurry after alladditions and prior to liquefaction was 31.4%. The canisters were thensealed with caps, and loaded into Lab-O-Mat. Liquefaction happened at85° C. for two hours with constant rotation.

After liquefaction done, the canisters were taken off from Lab-O-Mat andput on ice for fast cooling. The cooled corn mashes were then removedfrom canisters to beakers, and prepared for simultaneoussaccharification and fermentation (SSF). Each SSF treatment was run infive replicate. 3 ppm penicillin and 800 ppm urea was supplementedbefore SSF started, Approximately 5 g of each corn mash (liquefiedabove) was added to 15 ml polypropylene tube. The tubes were prepared bydrilling a 1/32 inch (1.5 mm) hole and the empty tubes were then weighedbefore corn slurry was added. The tubes were weighed again after mashwas added to determine the exact weight of mash in each tube. Each tubewas dosed with 0.6 AGU/gDS of Glucoamylase SA (GSA). Actual enzymedosages were based on the exact weight of corn slurry in each tube.After enzyme dosage, each tube was added with 100 ul of ETHANOL RED™yeast propagate, and then were incubated in 32° C. water bath for 54hrs. Samples were taken at the end of fermentation for HPLC analysis.The HPLC preparation consisted of stopping the reaction by addition of50 micro liters of 40% H₂SO₄, centrifuging, and filtering through a 0.45micrometer filter. Samples were stored at 4° C. until analysis. Agilent™1100 HPLC system coupled with RI detector was used to determine ethanoland oligosaccharides concentration. The separation column was aminexHPX-87H ion exclusion column (300 mm×7.8 mm) from BioRad™.

TABLE 20 Xylanase tested in liquefaction Source Family Action DSC, ° C.Talaromyces leycettanus GH10 Xylanase 89 (SEQ ID NO: 5 herein)Dictyoglomus thermophilum GH11 Xylanase 106 (SEQ ID NO: 34 herein)

TABLE 21 Enzyme dosage in liquefaction AA dose (KNU- EG dose Xyl dose(μg Liquefaction treatment A/g DS) (μg EP/gDS EP/gDS AA control 0.12AA + EG 0.12 100 AA + EG + Xyl 0.12 50 50 AA + Xyl 0.12 50

Results:

The ethanol yield from each treatment was summarized in Table 22 below.The TI xylanase has good performance by itself and on top of EG. By thesame enzyme dosage of 100 ug/gDS, the combination of xylanase and EGgave higher ethanol yield than EG itself. From GPC data afterliquefaction, it was found that the treatment with combination of TIxylanase and TI EG yielded smaller fragments than treatment with TI EGalone, indicating the TI xylanase was able to cut the corn fiber intosmaller fragments, hence help to release more bound starch.

TABLE 22 Summarized Ethanol Yield and Percent Change ResultsLiquefaction Treatment Ethanol (% w/v) Ethanol Increase (%) AA onlycontrol 13.40 — AA + EG100 13.50 0.7% AA + EG50 + Tl Xyl50 13.65 1.9%AA + Tl Xyl50 13.50 0.8% AA + EG50 + Dt Xyl50 13.55 1.1% AA + Dt Xyl5013.44 0.3%

Example 20

Adding Thermostable Hemicellulase in Liquefaction of Corn withAlpha-Amylase—Effect on Ethanol Yield

All treatments were evaluated via 18 g liquefaction. Ground corn flourwas obtained from an industrial corn ethanol plant to be used for theexperiment. The dry solids (% DS) of the corn flour was 86.5% asdetermined by Mettler-Toledo HB43 halogen moisture balance in duplicate.For liquefaction of the corn flour, 6.4±0.1 g corn flour was weighedinto a 40 ml Oak Ridge tube. Tap water was added to reach % DS of 32.2%and mass of 17.2±0.2 g corn slurry. The pH of the corn slurry was foundto be about 6.0; 23 μl of 40% H₂SO₄ was added to each tube to reduce thepH to 5.0 for liquefaction.

The alpha-amylase (AA369) was used. In addition to the AA-only control,six thermostable hemicellulases were evaluated as shown in Table 23.Each tube was dosed with the appropriate amount of diluted enzyme asshown in Table 24 below. Each liquefaction treatment was tested inquadruplicate along with six controls without hemicellulase added.Actual enzyme dosages were calculated based on the mass of corn slurryin each tube; tap water was added to bring the calculated % DS of alltubes to 31.0%. The final volume of the corn slurry after all additionsand prior to liquefaction was 18.1±0.4 g. After enzyme and wateraddition, the tubes were sealed tightly, shaken thoroughly, and thenplaced in a Boekel Big SHOT III hybridization oven Model 230402 with arack that holds and rotates the tubes vertically around a fixed axis.The oven was preheated to 80.0±0.5° C. as determined by a calibratedthermometer attached to the oven rack. The incubator temperaturedecreased while the door was open for loading; the temperature on thethermometer was monitored until it reached 75° C. at which time thetimer was started for a two hour incubation with rotation set at speed20 rpm.

TABLE 23 List of Hemicellulase Source, family and Sequence ID DSC Sourceof Hemicellulase Family Action (° C.) Sequence ID Dictyoglomusthermophilum GH10 Xylanase 99 (SEQ ID NO: 3 herein) Rasamsoniabyssochlamydoides GH10 Xylanase 96 (SEQ ID NO: 4 herein) Aspergiliusfumigatus GH10 Xylanase 89 (SEQ ID NO: 8 herein) Talaromyces emersoniiGH3 Beta- 78 (SEQ ID NO: 6 herein) xylosidase Dictyoglomus thermophilumGH11 Xylanase — (SEQ ID NO: 34 herein) Trichoderma reesei CE5Acetylxylan 69 (SEQ ID NO: 7 herein) esterase

TABLE 24 Enzyme Dosage in Liquefaction Hemicellulase dose (μgLiquefaction EP (Enzyme Protein)/g Treatments AA dose (KNU-A/g DS) DS)AA 0.12 AA + Hemicellulase 0.12 100

After incubating the tubes for two hours at 80° C., the tubes wereremoved from the hybridization oven and submerged in cool water withoccasional mixing for about 30 minutes until they were cool to thetouch. The caps were removed and a small spatula was used to pushmaterial stuck to the sides of the tubes down. Urea and penicillinsolutions prepared in-house were added to each tube to reach finalconcentrations of 700 ppm and 3 ppm, respectively. Each tube was dosedwith the appropriate amount of diluted Glucoamylase SA (GSA). Actualenzyme dosage was 0.77 AGU/g DS based on the average weight of liquefiedcorn mash in each tube; tap water was added as needed to bring the DS ofeach tube to 31%. All tubes had 400 μL of yeast rehydrated in tap wateradded; the volume is based on the average mash weight in each tube and ayeast dose of 20 μl/g corn mash rounded up to the nearest 100 μl. Thetubes were then covered with caps with septa, vented by inserting a 22gauge needle, and placed in an Infors HT MultitronPro humiditycontrolled incubator for Simultaneous Saccharification and Fermentation(SSF). The temperature was 32° C. and humidity was set at 80%; the tubeswere mixed by hand twice a day throughout fermentation.

Samples were collected for HPLC analysis after 65 hours of fermentation.This process began with stopping the enzyme and yeast reactions byadding 200 μL of 40% H₂SO₄ (10 μl/g corn mash, rounded up to the nearest100 μl) and mixing to distribute the acid; final calculated DS was 29.7%at the end of SSF. HPLC sample preparation consisted of transferringabout 5 g of fermented corn mash to a 15 ml Falcon tube, centrifuging at3000 rpm for 8 minutes, and passing the supernatant through a 0.45 μmfilter into an HPLC vial. Samples were stored at 4° C. until analysis.The system used to determine ethanol and oligosaccharides concentrationwas an Agilent™ 100 HPLC system coupled with an RI detector. Theseparation column was a BioRad™ Aminex HPX-87H ion exclusion column (300mm×7.8 mm).

Results

The final ethanol and percentage of ethanol increase by addition ofhemicellulase are summarized in Table 25 below.

TABLE 25 Summarized Ethanol Yield and Percent Change Results EthanolEthanol Liquefaction Treatment (% w/v) Increase (%) AA only control13.33 — AA + Dictyoglomus thermophilum GH10 13.59 2.0% xylanase AA +Rasamsonia byssochlamydoides 13.74 3.1% GH10 xylanase AA + Aspergillusfumigatus GH10 xylanase 13.47 1.1% AA + Talaromyces emersonii GH3 beta-13.29 −0.3% xylosidase AA + Dictyoglomus thermophilum GH11 13.39 0.5%xylanase AA + Trichoderma reesei CE5 acetylxylan 13.35 0.2% esterase

Example 21 Adding Thermostable Hemicellulase in Liquefaction ofCorn—Effect on Ethanol Yield

All treatments were evaluated via 18 g liquefaction. Ground corn flourwas obtained from an industrial corn ethanol plant to be used for theexperiment. The dry solids (% DS) of the corn flour was 86.5% asdetermined by Mettler-Toledo HB43 halogen moisture balance in duplicate.For liquefaction of the corn flour, 6.4±0.1 g corn flour was weighedinto a 40 ml Oak Ridge tube. Tap water was added to reach % DS of 31.5%and mass of 17.6±0.2 g corn slurry. The pH of the corn slurry was foundto be about 6.0; 23 μl of 40% H₂SO₄ was added to each tube to reduce thepH to 5.0 for liquefaction.

A thermostable hemicellulase from Talaromyces leycettanus (TI Xyl) wasevaluated as shown in Table 26. This hemicellulase was combined withalpha-amylase (AA369), thermostable glucoamylase (Po 498), thermostableprotease (Protease Pfu) as indicated in the Table 26 below to evaluatethe effect of the thermostable hemicellulase in liquefaction. Each tubewas dosed with the appropriate amount of diluted enzyme as shown inTable 26 below. Each liquefaction treatment was tested in triplicatealong with three controls without hemicellulase added. Actual enzymedosages were calculated based on the mass of corn slurry in each tube;tap water was added to bring the calculated % DS of all tubes to 31.0%.The final volume of the corn slurry after all additions and prior toliquefaction was 18.1±0.4 g. After enzyme and water addition, the tubeswere sealed tightly, shaken thoroughly, and then placed in a BoekelBoekel Big SHOT III hybridization oven Model 230402 with a rack thatholds and rotates the tubes vertically around a fixed axis. The oven waspreheated to 80.0±0.5° C. as determined by a calibrated thermometerattached to the oven rack. The incubator temperature decreased while thedoor was open for loading; the temperature on the thermometer wasmonitored until it reached 75° C. at which time the timer was startedfor a two-hour incubation with rotation set at speed 20 rpm.

Source of Enzyme Family Action Sequence ID Talaromyces leycettanus GH10Xylanase (SEQ ID NO: 5 herein)

TABLE 26 Enzyme Dosage in Liquefaction GA498 Protease Hemicellulase AA369 dose dose Pfu dose Tl xylanase Liquefaction (KNU-A/g (μg EP*/g (μgEP*/g dose (μg EP*/ Treatments DS) DS) DS) g DS) AA 0.12 — — — AA + GA0.12 4.5 — — AA + High 0.12 — 3.0 — Protease AA + 0.12 4.5 0.0385 —Protease + GA AA + High 0.12 4.5 3.0 — Protease + GA AA + 0.12 — — 100Hemicellulase AA + GA + 0.12 4.5 — 100 Hemicellulase AA + Protease +0.12 — 3.0 100 Hemicellulase AA + Protease + — — — 100 GA +Hemicellulase AA + Protease + — — — 100 GA + Hemicellulase *EP is enzymeprotein

After incubating the tubes for two hours at 80° C., the tubes wereremoved from the hybridization oven and submerged in cool water withoccasional mixing for about 30 minutes until they were cool to thetouch. The caps were removed and a small spatula was used to pushmaterial stuck to the sides of the tubes down. Urea and penicillinsolutions were added to each tube to reach final concentrations of 700ppm and 3 ppm, respectively. Each tube was dosed with the appropriateamount of diluted glucoamylase (GSA). Actual enzyme dosage was 0.6 AGU/gDS based on the average weight of liquefied corn mash in each tube; tapwater was added as needed to bring the DS of each tube to 31.2±0.1%. Alltubes had 400 μL of yeast rehydrated in tap water added; the volume isbased on the average mash weight in each tube and a yeast dose of 20μl/g corn mash rounded up to the nearest 100 μl. The tubes were thencovered with caps with septa, vented by inserting a 22 gauge needle, andplaced in an Infors HT MultitronPro humidity controlled incubator forSimultaneous Saccharification and Fermentation (SSF). The temperaturewas 32° C. and humidity was set at 80%; the tubes were mixed by handtwice a day throughout fermentation.

Samples were collected for HPLC analysis after 65 hours of fermentation.This process involved stopping the enzyme and yeast reactions by adding200 μL of 40% H₂SO₄ (10 μl/g corn mash, rounded up to the nearest 100μl) and mixing to distribute the acid; final calculated DS was 30±0.1%at the end of SSF. HPLC sample preparation consisted of transferringabout 5 g of fermented corn mash to a 15 ml Falcon tube, centrifuging at3000 rpm for 8 minutes, and passing the supernatant through a 0.45 μmfilter into an HPLC vial. Samples were stored at 4° C. until analysis.The system used to determine ethanol and oligosaccharides concentrationwas an Agilent™ 100 HPLC system coupled with an RI detector. Theseparation column was a BioRad™ Aminex HPX-87H ion exclusion column (300mm×7.8 mm).

Additionally, the samples were analysed for solubilized feruloyatedarabinoxylan (SFA) as an indication of hydrolysis of xylan in corn.Analysis was completed using a Tecan Safire V 2.20 08/02 plate readerusing XFLUOR4BETA Version: E 4.22d. To prepare samples for analysis, aportion of the 0.45 μm filtered supernatant was diluted eight-fold indeionized water. 200 μl of the resulting solution was transferred to aUV-compatible 96-well microtiter plate and A320 was measured; severalwells with 200 μl deionized water were included to use a blanks.

Results

The final ethanol and percentage of ethanol increase by addition ofhemicellulase to thermostable GA, thermostable protease and formulatedproducts in liquefaction are summarized in Table 27 below. Treatmentscontaining hemicellulase all showed at least 1% increase in ethanolyield over the same treatment without hemicelluase. The treatments withonly AA and AA+Protease+GA were found to have statistically higherethanol yield of 2.0% and 2.1%, respectively, when hemicellulase wasadded in liquefaction. Statistical analysis was completed usingDunnett's test for mean comparison (Statistics program used was SASJMP).

SFA and percentage of SFA increase by addition of hemicellulase on topof various activities and formulated products in liquefaction aresummarized in Table 27 below. Thermostable hemicellulase in liquefactionincreased SFA in supernatant over the same treatment withouthemicellulase in all cases.

TABLE 27 Summarized Ethanol Yield and Percent Change Over Same TreatmentWithout Hemicellulase Ethanol Liquefaction Treatment Ethanol (% w/v)Increase (%) AA 12.99 — AA + GA 13.13 — AA + High Protease 13.31 — AA +Protease + GA 13.07 — AA + High Protease + GA 13.40 — AA + Hemicellulase13.25 2.0% AA + GA + Hemicellulase 13.32 1.5% AA + Protease +Hemicellulase 13.52 1.5% AA + Protease + GA + Hemicellulase 13.35 2.1%AA + High Protease + GA + 13.58 1.3% Hemicellulase

TABLE 27 Summarized Solubilized Feruloyated Arabinoxylan (SFA) by A320Results and Percent Change Over Same Treatment Without HemicellulaseCorrected* Liquefaction Treatment Absorbance Percent Change AA 7.79 —AA + GA 7.8 — AA + Protease 7.52 — AA + Protease + GA 7.67 — AA + HighProtease + GA 7.95 — AA + Hemicellulase 8.33 7.0% AA + GA +Hemicellulase 8.07 3.5% AA + Protease + Hemicellulase 8.66 15.1% AA +Protease + GA + Hemicellulase 9.07 18.3% AA + High Protease + GA + 8.9112.0% Hemicellulase *Value corrected for eight-fold dilution and blanksubtracted.

Example 22 The Impact of Grind Size and Thermostable Xylanase Additionin Liquefaction—Effect on Ethanol Yield

All treatments were evaluated via 100 g small-scale liquefaction. Cornflour obtained from an industrial corn ethanol plant was used for theexperiment. To obtain the “Turkish ground” corn, the industrial cornflour was ground in-lab using a Bunn® Coffee Mill. A sieve analysis wasperformed on the industrial corn flour and Turkish ground corn flour. Toperform sieve analysis, 30 g of corn flour sample was weighed and placedin the Advantech Sonic Sifter. The sifter was run for 2 minutes usingthe sift/pulse setting at an amplitude of 8. Table 28 displays theresulting grind size distribution of the corn flour. The dry solids (DS)of the industrial ground corn was 86.48% and the Turkish ground corn was86.58%, as determined by Mettler-Toledo HB43 halogen moisture balance.Each of the three liquefaction treatments were run in triplicate in theLab-O-Mat (nine canisters total). Corn and tap water were added to eachcanister such that each corn slurry had a DS of 32%. The corn flourtype, corn weight and tap water weight added the canisters for eachtreatment is as shown in Table 29. The pH of each corn slurry wasadjusted to 5.0 using 40% H₂SO₄. Each canister was dosed with theappropriate amount of diluted enzyme as shown in Table 29. The totalweight of corn, tap water and diluted enzyme equaled 100 g. Thealpha-amylase (AA369) and the Dictyoglomus thermophilum GH11 xylanase(Dt xylanase from Table 20) were used. The canisters were then sealedwith caps and loaded into the Lab-O-Mat. Liquefaction happened at 80° C.for two hours with constant rotation.

After liquefaction was done, the canisters were taken off from theLab-O-Mat and put on ice for fast cooling. The cooled corn mashes werethen removed from canisters to beakers, and prepared for simultaneoussaccharification and fermentation (SSF). Five SSF replicates were runfor each canister, thus providing 15 total SSF replicates perliquefaction treatment. To all SSF corn mashes, 3 ppm of penicillin and1000 ppm of urea was added. Approximately 5 g of each corn mash(liquefied above) was added to a 15 ml polypropylene tube. The tubeswere prepared by drilling a 1/32 inch (1.5 mm) hole and empty tubes werethen weighed before liquefied corn mash was added. The tubes wereweighted again after mash was added to determine the exact weight ofmash in each tube. Each tube was dosed with 0.6 AGU/gDS of SPIRIZYMEEXCEL XHS. Actual enzyme dosages were based on the exact weight of thecorn mash in each tube. After enzyme dosage, 100 μl of ETHANOL RED™yeast propagate was added to each tube and tubes were then incubated ina 32° C. water bath for 54 hours. Samples were taken at the end offermentation for HPLC analysis. The HPLC preparation consisted ofstopping the reaction by addition of 50 μl of 40% H₂SO₄, centrifugingand filtering through a 0.45μ filter. Samples were stored at 4° C. untilanalysis. Agilent™ 1100 HPLC system couple with RI detector was used todetermine concentrations of ethanol and oligosaccharides. The separationcolumn was aminex HPX-87H ion exclusion column (300 mm×7.8 mm) fromBioRad™.

TABLE 28 Sieve Analysis. Turkish Sieve Industrial Ground Corn Size (μm)Corn Flour Flour 2000 0.5%  0.6% 1000 16.6%  6.7% 850 8.6%  9.1% 42535.5% 44.7% 250 11.2% 15.2% 180 5.5%  5.5% <180 13.7% 13.6%

TABLE 29 Summary of Contents in Liquefaction Canisters. Corn CornWeight/ Tap Water Xylanase Flour Canister Weight/Canister AA369 DoseDose Type (g) (g) (KNU(AH)/gDS) (μg EP/gDS) Industrial 37.00 62.70 0.120 Industrial 37.00 62.57 0.12 100 Turkish 36.96 62.61 0.12 100

Results:

The ethanol yield from each treatment is summarized in Table 30 below.The Dt xylanase provided a boost in ethanol when added to both cornflours. The increased ethanol yield of the Dt xylanase in combinationwith the industrial corn flour indicates that the Dt xylanase was ableto break down the corn fiber and thus help to release fiber-boundstarch, Dt xylanase in combination with Turkish ground industrial cornflour delivered the highest ethanol performance, indicating optimalperformance when fiber-bound starch release and further mechanicalgrinding of the corn flour are combined.

TABLE 30 Summarized Ethanol Yield and Percent Change Results. EthanolEthanol Increase Liquefaction Treatment (% w/v) (%) Industrial CornFlour + AA only 13.16 — Industrial Corn Flour + AA + Dt Xyl 13.33 1.3%Turkish Ground Corn Flour + AA + Dt 13.60 3.3% Xyl

1. A process for producing fermentation products from starch-containingmaterial comprising the steps of: i) liquefying the starch-containingmaterial at a temperature above the initial gelatinization temperatureusing: an alpha-amylase; a hemicellulase having a Melting Point (DSC)above 80° C.; ii) saccharifying using a carbohydrate-source generatingenzyme; iii) fermenting using a fermenting organism.
 2. The process ofclaim 1, wherein the hemicellulase is a xylanase.
 3. The process ofclaim 1, wherein the hemicellulase has a Melting Point (DSC) above 82°C., above 84° C., above 86° C., above 88° C., above 88° C., above 90°C., above 92° C., above 94° C., above 96° C., above 98° C., or above100° C.
 4. The process of claim 2, wherein the xylanase has at least60%, such as at least 70%, such as at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, even at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identity to the mature part of the polypeptide of SEQID NO: 3 herein or SEQ ID NO: 34 herein.
 5. The process of claim 2,wherein the xylanase has at least 60%, at least 70%, at least 75%identity, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to the maturepart of the polypeptide of SEQ ID NO: 4 herein.
 6. The process of claim2, wherein the xylanase has at least 60%, at least 70%, at least 75%identity, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to the maturepart of the polypeptide of SEQ ID NO: 5 herein.
 7. The process of claim1, wherein the xylanase has at least 60%, at least 70%, at least 75%identity, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to the maturepart of the polypeptide of SEQ ID NO: 8 herein.
 8. The process of claim1, wherein: an alpha-amylase; a hemicellulase having a Melting Point(DSC) above 80° C., above 85° C., above 90° C., or 95° C.; and anoptional endoglucanase having Melting Point (DSC) above 70° C., above75° C., above 80° C., or above 85° C.; are present and/or added inliquefaction step i).
 9. The process of claim 1 further comprises, priorto the liquefaction step i), the steps of: a) reducing the particle sizeof the starch-containing material; b) forming a slurry comprising thestarch-containing material and water.
 10. The process of claim 1,wherein: i) at least 50%, at least 70%, at least 80%, or at least 90% ofthe starch-containing material fit through a sieve with #6 screen; ii)at least 50%, at least 59% or 60%, of the starch-containing material fitthrough a sieve with a 0.25 to 0.425 mm screen; of iii) at least 75% ofthe starch-containing material fit through a sieve with less than a 0.5mm screen; or iv) at least 79% or 80% of the starch-containing materialfit through a sieve with equal to or less than a 0.425 mm screen. 11.The process of claim 1, wherein the pH during liquefaction is between4.0-6.5, 4.5-6.2, 4.8-6.0, or between 5.0-5.8.
 12. The process of claim1, wherein the temperature during liquefaction is in the range from70-100° C., between 70-95° C., between 75-90° C., or between 80-90° C.13. The process of claim 1, wherein a jet-cooking step is carried outbefore liquefaction in step i).
 14. The process of claim 13, wherein thejet-cooking is carried out at a temperature between 95-160° C., between110-145° C., 120-140° C., or 125-135° C., for about 1-15 minutes, or forabout 3-10 minutes.
 15. The process of claim 1, wherein saccharificationand fermentation is carried out sequentially or simultaneously.
 16. Theprocess of claim 1 wherein saccharification is carried out at atemperature from 20-75° C., from 40-70° C., and at a pH between 4 and 5.17. The process of claim 1, wherein fermentation or simultaneoussaccharification and fermentation (SSF) is carried out at a temperaturefrom 25° C. to 40° C., from 28° C. to 35° C., from 30° C. to 34° C., for6 to 120 hours, or 24 to 96 hours.
 18. The process of claim 1, whereinthe fermentation product is recovered after fermentation.
 19. Theprocess of claim 1, wherein the fermentation product is an alcohol. 20.The process of claim 1, wherein the starch-containing starting materialis whole grains.
 21. The process of claim 1, wherein thestarch-containing material is derived from corn, wheat, barley, rye,milo, sago, cassava, manioc, tapioca, sorghum, rice or potatoes.
 22. Theprocess of claim 1, wherein the fermenting organism is yeast.
 23. Theprocess of claim 1, further wherein a protease is present and/or addedin liquefaction.
 24. The process of claim 1, further wherein acarbohydrate-source generating enzyme, preferably a glucoamylase, ispresent and/or added during liquefaction step i).
 25. The process ofclaim 1, further wherein a pullulanase is present and/or added duringliquefaction and/or saccharification.
 26. The process of claim 1,further wherein an endoglucnase is present and/or added duringliquefaction step i).
 27. The process of claims 1-25 claim 1, wherein aphytase is present and/or added during liquefaction and/orsaccharification.
 28. A composition comprising: an alpha-amylase; ahemicellulase having a Melting Point (DSC) above 80° C., above 85° C.,above 90° C., or above 95° C.; optionally an endoglucanase; optionally aprotease; and optionally a carbohydrate-source generating enzyme,preferably a glucoamylase.
 29. The composition of claim 28, furthercomprising a pullulanase.
 30. The composition of claim 28, furthercomprising a phytase.
 31. (canceled)